U.S. patent number 7,981,420 [Application Number 10/451,586] was granted by the patent office on 2011-07-19 for therapeutic use of antibodies directed against repulsive guidance molecule (rgm).
This patent grant is currently assigned to Max-Planck-Gesellschaft Zur Foederung der Wissenschaften E.V.. Invention is credited to Jens S. Anderson, Friedrich Bonhoeffer, Paolo Macchi, Matthias Mann, Philippe P. Monnier, Bernhard K. Mueller, Bernd Stahl.
United States Patent |
7,981,420 |
Mueller , et al. |
July 19, 2011 |
Therapeutic use of antibodies directed against repulsive guidance
molecule (RGM)
Abstract
The present invention relates to the use of a modulator of a
polypeptide having or comprising an amino acid sequence as
disclosed herein or of a functional fragment or derivative thereof
or of a polynucleotide encoding said polypeptide or fragment or
derivative for the preparation of a pharmaceutical composition for
preventing, alleviating or treating diseases or conditions
associated with the degeneration or injury of vertebrate nervous
tissue, associated with seizures or associated with angiogenic
disorders or disorders of the cardio-vascular system. Furthermore,
the invention provides for the use of a modulator of a polypeptide
having or comprising said amino acid sequence of a functional
fragment or derivative thereof or of a polynucleotide encoding said
polypeptide or fragment or derivative for the preparation of a
pharmaceutical composition for preventing, alleviating or treating
diseases or conditions associated with the degeneration or injury
of vertebrate nervous tissue, associated with angiogenic disorders
or disorders of the cardio-vascular system. In addition the
invention provides for the use of said polypeptide or said
functional fragment or derivative thereof for the preparation of a
pharmaceutical composition for preventing or treating tumor growth
or formation of tumor metastases or as a marker of stem cells.
Inventors: |
Mueller; Bernhard K. (Neustadt,
DE), Monnier; Philippe P. (Toronto, CA),
Macchi; Paolo (Tubingen, DE), Bonhoeffer;
Friedrich (Tubingen, DE), Stahl; Bernd (Tubingen,
DE), Mann; Matthias (Odense M, DK),
Anderson; Jens S. (Odense SO, DK) |
Assignee: |
Max-Planck-Gesellschaft Zur
Foederung der Wissenschaften E.V. (Munich, DE)
|
Family
ID: |
8170794 |
Appl.
No.: |
10/451,586 |
Filed: |
December 21, 2001 |
PCT
Filed: |
December 21, 2001 |
PCT No.: |
PCT/EP01/15289 |
371(c)(1),(2),(4) Date: |
December 08, 2003 |
PCT
Pub. No.: |
WO02/051438 |
PCT
Pub. Date: |
July 04, 2002 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20040102376 A1 |
May 27, 2004 |
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Foreign Application Priority Data
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|
|
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Dec 22, 2000 [EP] |
|
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00128356 |
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Current U.S.
Class: |
424/141.1;
424/139.1 |
Current CPC
Class: |
C07K
14/465 (20130101); A61P 21/04 (20180101); A61P
9/10 (20180101); A61P 37/08 (20180101); A61P
35/00 (20180101); A61P 17/02 (20180101); A61P
31/04 (20180101); A61P 25/08 (20180101); A61P
25/14 (20180101); C07K 14/4702 (20130101); A61P
25/28 (20180101); A61K 38/1709 (20130101); A61P
9/00 (20180101); A61P 25/00 (20180101); A61P
25/16 (20180101); A61P 25/02 (20180101); A61P
43/00 (20180101); C07K 16/28 (20130101); A61P
13/12 (20180101); A61P 3/10 (20180101); A61P
39/02 (20180101); A61P 29/00 (20180101); A61P
37/00 (20180101); A61P 19/08 (20180101); C07K
14/47 (20130101); A61K 2039/505 (20130101) |
Current International
Class: |
A61K
39/395 (20060101); C07K 16/28 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO |
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WO |
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May 2006 |
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WO |
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WO |
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Other References
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Application to Dissect RGMa Activity on Axonal Outgrowth," The
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neural progenitor populations and migrating neuroblasts in the
embryonic mouse forebrain, Neuroscience (2006), 142(3): 703-16.
cited by other .
Muller, B.K., et al., "Chromophore-assisted laser inactivation of a
repulsive axonal guidance molecule," Current Biology 1996, vol. 6
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15, 2010. cited by other .
B. Mueller et al., "RGM, a repulsive guidance molecule, is involved
in retinal axon guidance in vitro." Taniguchi Symposia on Brain
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Form 1997, pp. 215-229. cited by other .
J. Frisen et al., "Ephrin-A5 (AL-1/RAGS) is essential for proper
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(English translation). cited by other.
|
Primary Examiner: Kemmerer; Elizabeth C.
Assistant Examiner: Ballard; Kimberly A.
Attorney, Agent or Firm: Mueller; Lisa V. Polsinelli
Shughart PC
Claims
The invention claimed is:
1. A method for stimulating nerve fiber growth following a spinal
cord injury, comprising inhibiting a polypeptide having an amino
acid sequence of SEQ ID NO: 20 by administering to a mammal in need
thereof an antibody that specifically binds to the amino acid
sequence of SEQ ID NO: 20.
2. The method of claim 1, wherein the mammal is a human.
3. The method of claim 1, wherein the mammal is a rat.
4. The method of claim 1, wherein the antibody is selected from the
group consisting of F3D4 and anti-RGM1.
5. The method of claim 4, wherein the antibody is F3D4.
6. The method of claim 4, wherein the antibody is anti-RGM1.
7. A method for stimulating nerve fiber growth following a brain
injury selected from the group consisting of ischemia and traumatic
brain injury, comprising inhibiting a polypeptide having an amino
acid sequence of SEQ ID NO: 20 by administering to a mammal in need
thereof an antibody that specifically binds to the amino acid
sequence of SEQ ID NO: 20.
8. The method of claim 7, wherein the mammal is a human.
9. The method of claim 7, wherein the mammal is a rat.
10. The method of claim 7, wherein the antibody is selected from
the group consisting of F3D4 and anti-RGM1.
11. The method of claim 10, wherein the antibody is F3D4.
12. The method of claim 10, wherein the antibody is anti-RGM1.
Description
This application is the United States national stage of
International Application No. PCT/EP01/15289, filed Dec. 21, 2001,
which was published under PCT Article 21(2) in English as
International Publication No. WO 02/051438, and which claims
benefit of European Patent Application No. 00128356.3 filed Dec.
22, 2000.
The present invention relates to the use of a modulator of a
polypeptide having or comprising an amino acid sequence as
disclosed herein or of a functional fragment or derivative thereof
or of a polynucleotide encoding said polypeptide or fragment or
derivative for the preparation of a pharmaceutical composition for
preventing, alleviating or treating diseases or conditions
associated with the degeneration or injury of vertebrate nervous
tissue, associated with angiogenic disorders or disorders of the
cardio-vascular system. Furthermore, the invention provides for the
use of a modulator of a polypeptide having or comprising said amino
add sequence or of a functional fragment or derivative thereof or
of a polynucleotide encoding said polypeptide or fragment or
derivative for the preparation of a pharmaceutical composition for
preventing, alleviating or treating diseases or conditions
associated with the degeneration or injury of vertebrate nervous
tissue, associated with seizures, associated with angiogenic
disorders or disorders of the cardiovascular system. In addition
the invention provides for the use of said polypeptide or said
functional fragment or derivative thereof for the preparation of a
pharmaceutical composition for preventing or treating tumor growth
or formation of tumor metastases or as a marker of stem cells.
Several documents are cited throughout the text of this
specification. The disclosure content of each of the documents
cited herein (including any manufacturer's specifications,
instructions, etc.) are hereby incorporated by reference.
The most important mechanism in formation of embryonic nervous
systems is the guidance of axons and growth cones by directional
guidance cues (Goodman, Annu. Rev. Neurosci. 19 (1996), 341-77;
Mueller, Annu. Rev. Neurosci 22, (1999), 351-88). A suitable model
system for studying this guidance process is the retinotectal
system of vertebrates. In the chick embryo approximately 2 million
retinal ganglion cell (RGC) axons leave each eye and grow towards
the contralateral tectum opticum to form a precise map (Mey &
Thanos, (1992); J. Himforschung 33, 673-702). Having arrived at the
anterior pole of the optic tectum, RGC axons start to invade their
tectal target to find their target neurons. Mapping occurs in such
a way, that RGC axons from nasal retina project to posterior tectum
and temporal axons to anterior tectum. Along the dorso-ventral
axis, axons coming from dorsal retina terminate in ventral tectum,
whereas those from ventral retina end up in dorsal tectum. In the
end a precise topographic map is formed, where neighborhood
relationships in the retina are preserved in the tectum, so that
axons from neighboring ganglion cells in the retina synapse with
neighboring tectal neurons. Most important for formation of this
map, are graded tectal guidance cues, read by retinal growth cones
carrying corresponding receptors which also show a graded
distribution (Sperry, Proc. Natl. Acad. Sci. USA 50 (1963),
703-710; Bonhoeffer & Gierer, Trends Neurosci. 7 (1984)
378-381). Position of each retinal growth cone in the tectal field
is therefore determined by two sets of gradients: receptor
gradients on ingrowing retinal axons and growth cones and ligand
gradients on tectal cells (Gierer, Development 101 (1987),
479-489). The existence of the graded tectal ligands has been
postulated from anatomical work, their identification however
proved to be extremely difficult and was only made possible with
the development of simple in vitro systems (Walter, Development 101
(1987), 685-96; Cox, Neuron 4 (1990), 31-7). In the stripe assay
RGC axons grow on a membrane carpet, consisting of alternating
lanes of anterior (a) and posterior (p) tectal membranes. On these
carpets, temporal retinal axons grow on anterior tectal membranes
and are repelled by the posterior lanes, whereas nasal axons do not
distinguish between a and p membranes (Walter, Development 101
(1987), 685-96). The same specificity is also observed in the
growth cone collapse assay (Raper & Kapfhammer, Neuron 4
(1990), 21-29), where temporal retinal growth cones collapse after
addition of posterior tectal membrane vesicles but do not react to
anterior tectal vesicles and where nasal growth cones are
insensitive to either type of vesicles (Cox, (1990), loc. cit.). In
both assay systems, treatment of posterior tectal membranes with
the enzyme phosphatidylinositol-specific phospholipase C (PI-PLC)
which cleaves the lipid anchor of glycosylphosphatidylinositol
(GPI)-linked proteins, removed their repellent and
collapse-inducing activity (Walter, J. Physiol 84 (1990),
104-10).
One of the first repulsive guidance molecules identified in the
retinotectal system of chick embryos was a GPI-anchored
glycoprotein with a molecular weight of 33/35 kDa (Stahl, Neuron 5
(1990), 735-43). This 33/35 kDa molecule, later termed RGM
(Repulsive Guidance Molecule), was active in both stripe and
collapse-assays and was shown to be expressed in a low-anterior
high-posterior gradient in the embryonic tecta of chick and rat
(Mueller, Curr. Biol. 6 (1996), 1497-502; Mueller, Japan Scientific
Societies Press (1997), 215-229). Due to the abnormal biochemical
behaviour of RGM, the precise amino acid sequence was not
obtainable. RGM was described as a molecule which is active during
vertebrate development. Interestingly, RGM is down-regulated in the
embryonic chick tectum after E12 and in the embryonic rat tectum
after P2 and completely disappears after the embryonic stages
(Muller (1992), Ph. D thesis University of Tubingen; Muller (1997)
Japan Scientific Societies, 215-229) In 1996, Muller (loc. cit)
have shown that CALI (chromophore-assisted laser inactivation) of
RGM eliminates the repulsive guidance activity of posterior tectal
membranes/RGM. However, due to the presence of other guidance
molecules, in particular of RAGS (repulsive axon guidance signal)
and ELF-1 (Eph ligand family 1), a complete elimination of guidance
was not always detected and it was speculated that RGM acts in
concert with RAGS (now termed ephrin-A5) and ELF-1 (ephrin-A2). It
was furthermore envisaged that RGM may be a co-factor potentiating
the activity of RAGS and ELF-1 in embryonic guidance events.
In 1980/81 the group of Aguayo found that, when peripheral neurons
are transplanted/grafted into injured CNS of adult, axon growth of
CNS neurons is induced (David, Science 214 (1981), 931-933).
Therefore, it was speculated that CNS neurons have still the
ability and capacity of neurite-outgrowth and/or regeneration, if a
suitable environment would be provided. Furthermore, it was
speculated that "CNS-neuron regeneration inhibitors" may exist.
In 1988, Caroni and Schwab (Neuron 1, 85-96) described two
inhibitiors of 35 kDa and 250 kDa, isolated from rat CNS myelin
(NI-35 and NI-250; see also Schnell, Nature 343 (1990) 269-272;
Caroni, J. Cell Biol. 106 (1988), 1291-1288).
In 2000, the DNA encoding for NI-220/250 was deduced and the
corresponding potent inhibitor of neurite growth was termed Nogo-A
(Chen, Nature 403 (2000), 434-438. The membrane-bound Nogo turned
out to be a member of the reticulon family (GrandPre, Nature 403
(2000), 439-444).
Further factors which mediate neuronal outgrowth inhibition have
first been isolated in grasshoppers, and termed "fasciclin IV" and
later "collapsin" in chicken. These inhibitors belong to the
so-called semaphorin family. Semaphorins have been reported in a
wide range of species and described as transmembrane proteins (see,
inter alia, Kolodkin Cell 75 (1993) 1389-99, Puschel, Neuron 14
(1995), 941-948). Yet, it was also shown that not all semaphorins
have inhibitory activity. Some members of said family, e.g.
semaphorin E, act as an attractive guidance signal for cortical
axons (Bagnard, Development 125 (1998), 5043-5053).
A further system of repulsive guidance molecules is the ephrin-Eph
system. Ephrins are ligands of the Eph receptor kinases and are
implicated as positional labels that may guide the development of
neural topographic maps (Flanagan, Ann. Rev. Neurosc. 21 (1998),
309-345). Ephrins are grouped in two classes, the A-ephrins which
are linked to the membrane by a glycosylphosphatidylinositol-anchor
(GPI-anchor) and the B-ephrins carrying a transmembrane domain (Eph
nomenclature committee 1997). Two members of the A-ephrins,
ephrin-A2 and ephrin-A5, expressed in low anterior-high posterior
gradients in the optic tectum, have recently been shown to be
involved in repulsive guidance of retinal ganglion cell axons in
vitro and in vivo (see, inter alia (Drescher, Cell 82 (1995),
359-70; Cheng, Cell 79 (1994), 157-168; Feldheim, Neuron 21 (1998),
563-74; Feldheim, Neuron 25 (2000), 563-74)
Considering the fact that a plurality of physiological disorders or
injuries are related to altered cellular migration processes, the
technical problems underlying the present invention was to provide
for means and methods for modifying altered developmental or
cellular (migration) processes which lead to disease
conditions.
Accordingly, the present invention relates to the use of an
modulator of a polypeptide having or comprising the amino acid
sequence of SEQ ID NOs.18, 20, 23 or 25 or of a functional fragment
or derivative thereof or of a polynucleotide encoding said
polypeptide or fragment or derivative for the preparation of a
pharmaceutical composition for preventing, alleviating or treating
diseases or conditions associated with the degeneration or injury
of vertebrate nervous tissue, associated with angiogenic disorders
or disorders of the cardiovascular system and associated with tumor
formation and tumor growth.
In context of the present invention, and as documented in the
appended examples, it was surprisingly found that the repulsive
guidance molecule (RGM) is not only expressed during vertebrate
development but is re-expressed in adult tissue, in particular in
damaged adult tissues. It was, inter alia, surprisingly found that
RGM is re-expressed after damage of the nervous tissue, after
traumatic events or focal ischemias. The present invention provides
for the complete nucleotide sequence and/or amino acid of RGM (see,
e.g. SEQ ID NO: 17 or 18 depicting the RGM sequence of chicken or
SEQ ID NO: 20 to 25 depicting the human RGM homologues.) RGM, as
pointed out herein above, is a glycoprotein, linked to membranes by
a GPI-anchor. Said GPI-anchor also carries a cross-reacting
determinant (CRD) epitope and its carbohydrate part is able to bind
peanut lectin. As documented herein, the RGM protein is a potent
growth inhibitor and can assert neurite growth inhibition in
picomolor concentrations.
The term "modulator" as employed herein relates to "inhibitors" as
well as "activators" of RGM function. Most preferably said
"modulation" is an inhibition, wherein said inhibition may be a
partial or a complete inhibition.
The term "amino acid sequence of SEQ ID NO: 18, 20, 23 or 25 as
employed herein relates to the amino acid sequence of RGM
(repulsive guidance molecule) and relates to the RGM polypeptide of
chicken or human, respectively. In particular, SEQ ID NOs: 20 and
21 depict human RGM1. Human RGM1 has been localized on chromosome
15. Further, human RGMs comprise RGM2 and RGM3. RGM2 is depicted in
SEQ ID NO: 23 (amino acid sequence) and is encoded by a nucleotide
sequence as shown in SEQ ID NO: 22. Human RGM2 has been localized
on chromosome 5. Furthermore, human RGM3 is shown in appended SEQ
ID NO: 25 (amino acid sequence) and encoded by a nucleotide
sequence as depicted in SEQ ID NO: 24. Human RGM3 is located on
chromosome 1. Yet, as will be discussed herein below, said term
relates also to further RGM homologues.
The term "(poly)peptide" means, in accordance with the present
invention, a peptide, a protein, or a (poly)peptide which
encompasses amino acid chains of a given length, wherein the amino
acid residues are linked by covalent peptide bonds. However,
peptidomimetics of such RGM proteins/(poly)peptides wherein amino
acid(s) and/or peptide bond(s) have been replaced by functional
analogs are also encompassed by the invention.
The present invention is not restricted to RGM from human, mouse or
chicken and its inhibitors but also relates to the use of
inhibitors of RGM or of RGM itself (or functional fragments or
derivatives thereof) from other species. Since the present
invention provides for the use of amino acid seuqences/polypeptides
of RGM and its corresponding inhibitors and since the amino acid
sequences of human and chicken RGM are disclosed herein, the person
skilled in the art is provided with the information to obtain RGM
sequences from other species, like, inter alia, mouse, rat, pig,
etc. The relevant methods are known in the art and may be carried
out by standard methods, employing, inter alia, degenerate and non
degenerate primers in PCR-techniques. Such molecular biology
methods are well known in the art and, e.g., described in Sambrook
(Molecular Cloning; A Laboratory Manual, 2.sup.nd Edition, Cold
Spring Harbour Laboratory Press, Cold Spring Harbour, N.Y. (1989))
and Ausubel, "Current Protocols in Molecular Biology", Green
Publishing Associates; and Wiley Interscience, N.Y. (1989).
Furthermore, as employed in the context of the present invention,
the term "RGM", "RGM modulator" and "RGM-inhibitor" also relates to
RGM molecules (and their corresponding inhibitors) which are
variants or homologs of the RGM molecules (and their inhibitors) as
described herein. "Homology" in this context is understood to refer
in this context to a sequence identity of RGMs of at least 70%,
preferably more than 80% and still more preferably more than 90% on
the amino acid level. The present invention, however, comprises
also (poly)peptides deviating from wildtype amino acid sequences of
human or chicken RGMs described herein, wherein said deviation may
be, for example, the result of amino acid and/or nucleotide
substitution(s), deletion(s), addition(s), insertion(s),
duplication(s), inversion(s) and/or recombination(s) either alone
or in combination. Those deviations may naturally occur or be
produced via recombinant DNA techniques well known in the art. The
term "variation" as employed herein also comprises "allelic
variants". These allelic variations may be naturally occurring
allelic variants, splice variants as well as synthetically produced
or genetically engineered variants.
The term "polynucleotide" in accordance with the present invention
comprises coding and, wherever applicable, non-coding sequences
(like promotors, enhancers etc.). It comprises DNA, RNA as well as
PNA. In accordance with the present invention, the term
"polynucleotide/nucleic acid molecule" comprises also any feasible
derivative of a nucleic acid to which a nucleic acid probe may
hybridize. Said nucleic acid probe itself may be a derivative of a
nucleic acid molecule capable of hybridizing to said nucleic acid
molecule or said derivative thereof. The term "nucleic acid
molecule" further comprises peptide nucleic acids (PNAs) containing
DNA analogs with amide backbone linkages (Nielsen, Science 254
(1991), 1497-1500). The term "nucleic acid molecule" which encodes
a RGM (poly)peptide or a functional fragment/derivative thereof, in
connection with the present invention, is defined either by (a) the
specific nucleic acid sequences encoding said (poly)peptide
specified in the present invention or (b) by nucleic acid sequences
hybridizing under stringent conditions to the complementary strand
of the nucleotide sequences of (a) and encoding a (poly)peptide
deviating from the nucleic acid of (a) by one or more nucleotide
substitutions, deletions, additions or inversions and wherein the
nucleotide sequence shows at least 70%, more preferably at least
80% identity with the nucleotide sequence of said encoded RGM
(poly)peptide having an amino acid sequence as defined herein above
and functions as a RGM (or a functional fragment/derivative
thereof).
The term "modulator" as employed herein also comprises the term
"inhibitor", as mentioned herein above.
The term "inhibitor of a polypeptide having or comprising the amino
acid sequence of SEQ ID NOs 18, 20, 23 or 25 or a functional
fragment or derivative thereof", therefore, not only relates to the
specific inhibitors of human or chicken RGM but also relates to
inhibitors of RGM (or functional fragments or derivatives thereof
of other species. Useful inhibitors are disclosed herein as well as
described herein below and in the appended examples.
The term "inhibitor" also comprises "modulators" of the RGM
polypeptides and/or the RGM encoding nucleic acid molecule/gene. In
context of this invention it is also envisaged that said
"modulation" leads, when desired, to an activation of RGM.
The term "functional fragment or derivative thereof" in context of
the present invention and in relation to the herein described RGM
molecules comprises fragments of the RGM molecules defined herein
having a length of at least 25, more preferably at least 50, more
preferably at least 75, even more preferably at least 100 amino
acids. Functional fragments of the herein identified RGM molecules
or RGM molecules of other species (homologous RGMs) may be
comprised in fusion and/or chimeric proteins. "Functional
fragments" comprise RGM fragments (or its encoding nucleic acid
molecules) which are able to replace RGM full length molecules in
corresponding assays (as disclosed herein in the appended examples,
e.g. collapse and/or stripe assays) or may elucidate an anti-RGM
specific immune-response and/or lead to specific anti-RGM
antibodies. An example of such a "functional fragment" is, inter
alia, the functional fragment of chicken RGM depicted in SEQ ID NO:
19. In context of the present invention, polynucleotides encoding
functional fragments of RGM and/or its derivatives have preferably
at least 15, more preferably at least 30, more preferably at least
90, more preferably of at least 150, more preferably of at least
300 nucleotides. The term "derivative" means in context of their
invention derivatives of RGM molecules and/or their encoding
nucleic acid molecules and refer to natural derivatives (like
allelic variants) as well as recombinantly produced
derivatives/variants which may differ from the herein described RGM
molecules by at least one modification/mutation, e.g. at least one
deletion, substitution, addition, inversion or duplication. The
term "derivative" also comprises chemical modifications. The term
"derivative" as employed herein in context of the RGM molecule also
comprises soluble RGM molecules which do not comprise any membrane
anchorage.
As mentioned herein above, the present invention provides for the
use of a modulator, preferably an inhibitor, of RGM molecules
and/or their corresponding encoding polynucleotides/nucleic acid
molecules for the preparation of a pharmaceutical composition for
preventing, alleviating or treating various disorders of the
nervous system, angiogenic disorders or disorders of the
cardiovascular system and malignancies of different etiology.
In a preferred embodiment, said disorders of the nervous system
comprise degeneration or injury of vertebrate nervous tissue, in
particular neurodegenerative diseases, nerve fiber injuries and
disorders related to nerve fiber losses.
Said neurodegenerative diseases may be selected from the group
consisting of motorneuronal diseases (MND), amyotrophic lateral
sclerosis (ALS), Alzheimers disease, Parkinsons disease,
progressive bulbar palsy, progressive muscular atrophy, HIV-related
dementia and spinal muscular atrophy(ies), Down's Syndrome,
Huntington's Disease, Creutzfeldt-Jacob Disease,
Gerstmann-Straeussler Syndrome, kuru, Scrapie, transmissible mink
encephalopathy, other unknown prion diseases, multiple system
atrophy, Riley-Day familial dysautonomia said nerve fiber injuries
may be selected from the group consisting of spinal cord
injury(ies), brain injuries related to raised intracranial
pressure, trauma, secondary damage due to increased intracranial
pressure, infection, infarction, exposure to toxic agents,
malignancy and paraneoplastic syndromes and wherein said disorders
related to nerve fiber losses may be selected from the group
consisting of paresis of nervus facialis, nervus medianus, nervus
ulnaris, nervus axillaris, nervus thoracicus longus, nervus
radialis and for of other peripheral nerves, and other aquired and
non-aquired deseases of the (human) central and peripheral nervous
system.
The above mentioned spinal cord and brain injuries not only
comprise traumatic injuries but also relate to injuries caused by
stroke, ischemia and the like. It is in particular envisaged that
the inhibitors as defined herein below and comprising, inter alia,
anti-RGM antibodies be employed in the medical art to stimulate
nerve fiber growth in individuals, in particular in vertebrates,
most preferably in humans.
In a more preferred embodiment of the present invention, the
invention provides for the use of a modulator, preferably an
inhibitor to RGM (or a functional fragment or derivative thereof)
for the preparation of a pharmaceutical composition for the
treatment of disorders of the cardiovascular system, wherein these
disorders, e.g., comprise disorders of the blood-brain barrier,
brain oedema, secondary brain damages due to increased intracranial
pressure, infection, infarction, ischemia, hypoxia, hypoglycemia,
exposure to toxic agents, malignancy, paraneoplastic syndromes.
It is envisaged, without being bound by theory, that RGM inhibitors
may stimulate surviving neurons to project collateral fibers into
the diseased tissue, e.g. the ischemic tissue.
As illustrated in the appended examples, RGM is expressed locally
at the side of artificial transection of brain/spinal cord tissue
in test animals (like rats), e.g., in the penumbra region
surrounding an ischemic core of a human suffering focal ischemia in
the temporal contex. Furthermore, it is documented in the appended
examples that RGM is, surprisingly, expressed in tissue(s) having
experienced from traumatic brain injuries. The invention also
relates to the use of a RGM polypeptide or a functional fragment or
derivative thereof or the use of a polynucleotide encoding the same
(polypeptides and polynucleotides as defined herein), wherein the
above described disease or condition associated with seizures is
epilepsy. An epilepsy is thereby characterized by an epileptic
seizure as a convulsion or transient abnormal event experienced by
the subject, e.g. a human patient, due to a paroxysmal discharge of
(cerebral) neurons. The epileptic seizures comprise tonic seizures,
tonic-clonic seizures (grand mal), myoclonic seizures, absence
seizures as well as akinetic seizures. Yet, also comprised are in
context of this invention simple partial seizures, e.g. Jacksonian
seizures and seizures due to perinatal trauma and/or fetal anoxia.
As mentioned herein below, the uses described herein relate in
particular to the preparation of pharmaceutical compositions for
the treatment of diseases/conditions associated with aberrant
sprouting of nerve fibres, like epilepsy; see also Routbort,
Neuroscience 94 (1999), 755-765.
In a even more preferred embodiment of the invention, the
modulator, preferably the inhibitor of RGM (or of its functional
fragment or derivative thereof or of its encoding nucleid acid
molecule) is used for the preparation of a pharmaceutical
composition for the modification of neovascularization. Said
modification may comprise activation as well as stimulation. It is
in particular envisaged that said neovascularisation be stimulated
and/or activated in diseased tissue, like inter alia, ischemic
and/or infarctious tissue. Furthermore, it is envisaged that the
RGM-inhibitors described herein may be employed in the regulation
of the blood-brain barrier permeability.
It is furthermore envisaged that said modulators, preferably said
inhibitors for RGM be employed in the alleviation, prevention
and/or inhibition of progression of vascular plaque formation (e.g.
artherosclerosis) in cardiovascular, cerebo-vascular and/or
nephrovascular diseases/disorders.
Furthermore, the present invention provides for the use of a
modulator, preferably an inhibitor of RGM as defined herein for the
preparation of a pharmaceutical composition for remyelination.
Therefore, the present invention provides for a pharmaceutical
composition for the treatment of demyelinating diseases of the CNS,
like multiple sclerosis or of demyelinating diseases like
peripheral neuropathy caused by diphteria toxin,
Landry-Guillain-Barre-Syndrom, Elsberg-Syndrom, Charcot-Marie-Tooth
disease and other polyneuropatias. A particular preferred inhibitor
of RGM in this context is an antibody directed against RGM, e.g. an
IgM antibody. It has previously be shown that certain IgMs bind to
oligodendrocytes and thereby induce remyelination. IgM antibodies
against RGM are known in the art and comprise e.g. the F3D4
described in the appended examples.
In addition the invention provides for the use of a RGM polypeptide
as defined herein or of a functional fragment or derivative thereof
or of a polynucleotide encoding said polypeptide or fragment or
derivative for the preparation of a pharmaceutical composition for
preventing, alleviating or treating diseases or conditions
associated with the activity of autoreactive immune cells or with
overactive inflammatory cells. Most preferably these cells are
T-cells.
Furthermore, the present invention relates to the use of a
modulator, preferably an inhibitor or another RGM binding molecule
of a RGM polypeptide or of a functional fragment or derivative
thereof or of a polynucleotide encoding said polypeptide or of
fragment/derivative thereof for modifying and/or altering the
differentiation status of neuronal stem cells and/or their
progenitors. Said stem cells are normally found in the
subventricular zones of many brain regions. It is known that
factors in the microenvironment of the brain dramatically influence
the differentiation of undifferentiated stem cells. It is assumed
that due to the characteristic expression of RGM in the
subventricular layers of many different brain regions, this
molecule could be a marker for stem cells. Furthermore, RGM
inhibitors, like antibodies could be useful markers for stem cells.
Most important in stem cell biology is the understanding of factors
influencing their differentiation. It is therefore assumed that RGM
inhibitors change the developmental fate of these cells.
As documented in the appended examples, RGM is not only expressed
in ischemic tissue but is also expressed in scar tissue surrounding
(brain) lesions.
It is particularly preferred that the modulator, preferably the
inhibitior of the RGM molecule (or its functional fragment or
derivative) is an antibody or a fragment or a derivative thereof,
is an aptamer, is a specific receptor molecule capable of
interacting with a RGM polypeptide or with a functional fragment or
derivative thereof, or is a specific nucleic acid molecule
interacting with a polynucleotide encoding an RGM and/or the
polypeptide as defined herein.
The antibody to be used in context of the present invention can be,
for example, polyclonal or monoclonal antibodies. Techniques for
the production of antibodies are well known in the art and
described, e.g. in Harlow and Lane "Antibodies, A Laboratory
Manual", CSH Press, Cold Spring Harbor, 1988. The production of
specific anti-RGM antibodies is further known in the art (see, e.g.
Muller (1996) loc.cit.) or described in the appended examples.
The term "antibody" as employed herein also comprises chimeric,
single chain and humanized antibodies, as well as antibody
fragments, like, inter alia, Fab fragments.
Antibody fragments or derivatives further comprise F(ab').sub.2, Fv
or scFv fragments; see, for example, Harlow and Lane, loc.cit.
Various procedures are known in the art and may be used for the
production of such antibodies and/or fragments, see also appended
examples. Thus, the (antibody) derivatives can be produced by
peptidomimetics. Further, techniques described for the production
of single chain antibodies (see, inter alia, U.S. Pat. No.
4,946,778) can be adapted to produce single chain antibodies to
polypeptide(s) of this invention. Also, transgenic animals may be
used to express humanized antibodies to polypeptides of this
invention. Most preferably, the antibody to be used in the
invention is a monoclonal antibody, for example the F3D4 antibody
(produced by a mouse hybridoma cell line deposited with
DSMZ-Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH,
Inhoffenstr. 7B, D-38124 Braunschweig, Germany, on May 25, 2010,
and assigned Accession No. DSM ACC3065) described in the appended
examples may be employed when an IgM is desired. The general
methodology for producing, monoclonal antibodies is well-known and
has been described in, for example, Kohler and Milstein, Nature 256
(1975), 494-496 and reviewed in J. G. R. Hurrel, ed., "Monoclonal
Hybridoma Antibodies Techniques and Applications", CRC Press Inc.,
Boco Raron, Fla. (1982), as well as that taught by L. T. Mimms et
al., Virology 176 (1990), 604-619.
Preferably, said antibodies (or inhibitors) are directed against
functional fragments of the RGM polypeptide. As pointed out herein
above and as documented in the appended examples, such functional
fragments are easily deducible for the person skilled in the art
and, correspondingly, relevant antibodies (or other inhibitors) may
be produced.
The "modulator", preferably the "inhibitor" as defined herein may
also be an aptamer. In the context of the present invention, the
term "aptamer" comprises nucleic acids such as RNA, ssDNA
(ss=single stranded), modified RNA, modified ssDNA or PNAs which
bind a plurality of target sequences having a high specificity and
affinity. Aptamers are well known in the art and, inter alia,
described in Famulok, Curr. Op. Chem. Biol. 2 (1998), 320-327. The
preparation of aptamers is well known in the art and may involve,
inter alia, the use of combinatorial RNA libraries to identify
binding sites (Gold, Ann. Rev. Biochem. 64 (1995), 763-797). Said
other receptors may, for example, be derived from said antibody
etc. by peptdomimetics.
Other specific "receptor" molecules which may function as
inhibitors of the RGM polypeptides are also comprised in this
invention. Said specific receptors may be deduced by methods known
in the art and comprise binding assays and/or interaction assays.
These may, inter alia, involve assays in the ELISA-format or
FRET-format. Said "inhibitor" may also comprise specific peptides
binding to and/or interfering with RGM.
Furthermore, the above recited "modulator", preferably "inhibitor"
may function at the level of RGM gene expression. Therefore, the
inhibitor may be a (specific) nucleic acid molecule interacting
with a polynucleotide encoding a RGM molecule (or a functional
fragment or derivative thereof.) These inhibitors may, e.g.,
comprise antisense nucleic acid molecules or ribozymes.
The nucleic acid molecule encoding RGM (and as disclosed herein,
e.g., SEQ ID NO: 17) may be employed to construct appropriate
anti-sense oligonucleotides. Said anti-sense oligonucleotides are
able to inhibit the function of wild-type (or mutant) RGM genes and
comprise, preferably, at least 15 nucleotides, more preferably at
least 20 nucleotides, even more preferably 30 nucleotides and most
preferably at least 40 nucleotides.
In addition, ribozyme approaches are also envisaged for use in this
invention. Ribozymes may specifically cleave the nucleic acid
molecule encoding RGMs.
In the context of the present invention ribozymes comprise, inter
alia, hammerhead ribozymes, hammerhead ribozymes with altered core
sequences or deoxyribozymes (see, e.g., Santoro, Proc. Natl. Acad.
Sci. USA 94 (1997), 4262) and may comprise natural and in vitro
selected and/or synthesized ribozymes. Nucleic acid molecules
according to the present invention which are complementary to
nucleic acid molecules coding for proteins/(poly)peptides
regulating, causing or contributing to obesity and/or encoding a
mammalian (poly)peptide involved in the regulation of body weight
(see herein below) may be used for the construction of appropriate
ribozymes (see, e.g., EP-B1 0 291 533, EP-A1 0 321 201, EP-A2 0 360
257) which specifically cleave nucleic acid molecules of the
invention. Selection of the appropriate target sites and
corresponding ribozymes can be done as described for example in
Steinecke, Ribozymes, Methods in Cell Biology 50, Galbraith, eds.
Academic Press, Inc. (1995), 449-460.
Said "inhibitor" may also comprise double-stranded RNAs, which lead
to RNA-mediated gene interference (see Sharp, Genes and Dev. 13
(1999), 139-141)
Further potential inhibitors of RGM may be found and/or deduced by
interaction assay and employing corresponding read-out systems.
These are known in the art and comprise, inter alia, two hybrid
screenings (as, described, inter alia, in EP-0 963 376, WO
98/25947, WO 00/02911) GST-pull-down columns, co-precipitation
assays from cell extracts as described, inter alia, in
Kasus-Jacobi, Oncogene 19 (2000), 2052-2059, "interaction-trap"
systems (as described, inter alia, in U.S. Pat. No. 6,004,746)
expression cloning (e.g. lambda gtII), phage display (as described,
inter alia, in U.S. Pat. No. 5,541,109), in vitro binding assays
and the like. Further interaction assay methods and corresponding
read out systems are, inter alia, described in U.S. Pat. No.
5,525,490, WO 99/51741, WO 00/17221, WO 00/14271 or WO
00/05410.
A further objective of the present invention is to provide for the
use of a RGM polypeptide and/or of polypeptide having or comprising
the amino acid sequence of SEQ ID NOs. 18, 20, 23 or 25 or of a
functional fragment or derivative thereof or of a polynucleotide
encoding said polypeptide or fragment or derivative for the
preparation of a pharmaceutical composition for preventing,
alleviating or treating diseases or conditions associated with
excessive collateral sprouting of nerve fibres.
The present invention, therefore, provides for the medical use of
RGM protein(s) and/or functional fragments/derivatives thereof or
for the use of polynucleotides encoding said RGM protein(s) in
conditions where excessive collateral sprouting occurs. Said
conditions comprise, but are not limited to, epilepsy, phantom pain
and neuropathic pain. For example, McNamara (Nat. Suppl. 399
(1999), A15-A22) has described that said sprouting occurs in
certain types of epilepsy. The RGM molecule, either naturally
isolated or recombinantly produced, or its functional
fragments/derivatives may therefore be employed as potent "stop"
signals for growing nerve fibres. The feasibility of such an
approach has been shown by Tanelian (Nat. Med. 3 (1997), 1398-1401)
who employed a semaphorin for inhibition of nerve fiber growth.
In yet another embodiment, the present invention provides for the
use of RGM and/or of a polypeptide having or comprising the amino
acid sequence of SEQ ID NOs 18, 20, 23 or 25 or of a functional
fragment or derivative thereof or of a polynucleotide encoding said
polypeptide or fragment or derivative for the preparation of a
pharmaceutical composition for preventing or treating tumor growth
or formation of tumor metastases.
RGM (naturally isolated or recombinantly produced) and/or
functional fragments thereof may be employed for the preparation of
a pharmaceutical composition for the treatment of neoplastic
disorders, in particular of disorders related to tumor (cell)
migration, metastasis and/or tumor invasion. Furthermore, it is
envisaged that RGM inhibits undesired neovascularisation. Said
neovascularisation, as an angiogenic disorder during neoplastic
events, should be prevented in order to limit, inter alia, tumor
growth.
Growth cones of neurons and (invasive) tumor cells secrets a
cocktail of proteases (uPA, tPA, MNPs, etc.) in order to degrade
extracellular matrix. Furthermore, similar mechanisms for adhesion
and (cell) migration are employed by these cellular systems.
RGM and/or its functional fragments may be employed to actively
stimulate withdrawal of lamellipodia of tumor cells and/or to
induce their collapse. As demonstrated in the appended examples RGM
also influences tumor growth behaviour, i.e. is capable of
negatively influencing tumor growth.
In addition the invention provides for the use of a RGM polypeptide
as defined herein or of a functional fragment or derivative thereof
or of a polynucleotide encoding said polypeptide or fragment or
derivative for the preparation of a pharmaceutical composition for
preventing, alleviating or treating diseases or conditions
associated with the activity of autoreactive immune cells or with
overactive inflammatory cells. Most preferably these cells are
T-cells.
In yet another embodiment, the invention provides for the use of a
RGM polypeptide having or comprising, inter alia, the amino acid
sequence of SEQ ID NOs.18, 20, 23 or 25 or of a functional fragment
or derivative thereof or of a polynucleotide encoding said
polypeptide or fragment or derivative for the preparation of a
pharmaceutical composition for the treatment of inflammation
processes and/or allergies, for wound healing or for the
suppression/alleviation of scar formation. Scar tissue is formed by
invading cells, most importantly by fibroblasts and/or glial cells.
Migration and adhesion of these cells are required to get to the
lesion side. RGM or an active fragment/derivative could prevent
accumulation of these cells in the lesion side, thereby preventing
or slowing down scar formation. In inflammatory reactions cells
migrate to the inflamed region and RGM or its active
fragment/derivative prevent or reduce migration of these cells to
the side of inflammation, thereby preventing overactive
inflammatory reactions.
In context of the present invention, the term "pharmaceutical
composition" also comprises optionally further comprising an
acceptable carrier and/or diluent and/or excipient. The
pharmaceutical composition of the present invention may be
particularly useful in preventing and/or treating pathological
disorders in vertebrates, like humans. Said pathological disorders
comprise, but are not limited to, neurological, neurodegenerative
and/or neoplastic disorders as well as disorders associated with
seizures, e.g. epilepsy. These disorders comprise, inter alia,
Alzheimer's disease, Parkinson's disease, amyotrophic lateral
sclerosis (FALS/SALS), ischemia, stroke, epilepsy, AIDS dementia
and cancer. The pharmaceutical composition may also be used for
prophylactic purposes. Examples of suitable pharmaceutical carriers
are well known in the art and include phosphate buffered saline
solutions, water, emulsions, such as oil/water emulsions, various
types of wetting agents, sterile solutions etc. Compositions
comprising such carriers can be formulated by well known
conventional methods. These pharmaceutical compositions can be
administered to the subject at a suitable dose. Administration of
the suitable compositions may be effected by different ways, e.g.,
by intravenous, intraperitoneal, subcutaneous, intramuscular,
topical, intradermal, intranasal or intrabronchial administration.
However, it is also envisaged that the pharmaceutical compositions
are directly applied to the nervous tissue. The dosage regimen will
be determined by the attending physician and clinical factors. As
is well known in the medical arts, dosages for any one patient
depends upon many factors, including the patient's size, body
surface area, general health, age, sex, the particular compound to
be administered, time and route of administration, and other drugs
being administered concurrently. Pharmaceutically active matter may
be present preferably, inter alia, in amounts between 1 ng and 1000
mg per dose, more preferably in amounts of 1 ng to 100 mg however,
doses below or above this exemplary range are envisioned,
especially considering the aforementioned factors. If the regimen
is a continuous infusion, it should also be in the range of 1 .mu.g
to 10 mg units per kilogram of body weight per minute,
respectively. Progress can be monitored by periodic assessment. The
compositions of the invention may be administered locally or
systemically. Administration will generally be parenterally, e.g.,
intravenously. The compositions of the invention may also be
administered directly to the target site, e.g., by biolistic
delivery to an internal or external target site or by catheter to a
site in an artery. Preparations for parenteral administration
include sterile aqueous or non-aqueous solutions, suspensions, and
emulsions. Examples of non-aqueous solvents are propylene glycol,
polyethylene glycol, vegetable oils such as olive oil, and
injectable organic esters such as ethyl oleate. Aqueous carriers
include water, alcoholic/aqueous solutions, emulsions or
suspensions, including saline and buffered media. Parenteral
vehicles include sodium chloride solution, Ringer's dextrose,
dextrose and sodium chloride, lactated Ringer's, or fixed oils.
Intravenous vehicles include fluid and nutrient replenishers,
electrolyte replenishers (such as those based on Ringer's
dextrose), and the like. Preservatives and other additives may also
be present such as, for example, antimicrobials, anti-oxidants,
chelating agents, and inert gases and the like. Furthermore, the
pharmaceutical composition of the invention may comprise further
agents, depending on the intended use of the pharmaceutical
composition. Such agents may be drugs acting on the central nervous
system as well as on small, unmyelinated sensory nerve terminals
(like in the skin), neurons of the peripheral nervous system of the
digestive tract, etc.
It is also understood that the pharmaceutical composition as
defined herein may comprise nucleic acid molecules encoding RGMs
(and/or functional fragments or derivatives thereof) or
corresponding RGM inhibitors or defined herein. As mentioned
herein-above, said inhibitors comprise, but are not limited to,
antibodies, aptamer, RGM-interacting peptides as well as inhibitors
interacting with the RGM-encoding polynucleotides.
Accordingly, the present invention also provides for a method of
treating, preventing and/or alleviating pathological disorders and
conditions as defined herein, whereby said method comprises
administering to a subject in need of such a treatment a
pharmaceutical composition/medicament as defined herein.
Preferably, said subject is a human.
The nucleic acid molecules may be particularly useful in gene
therapy approaches and may comprise DNA, RNA as well as PNA. Said
nucleic acid molecules may be comprised in suitable vectors, either
inter alia, gene expression vectors. Such a vector may be, e.g., a
plasmid, cosmid, virus, bacteriophage or another vector used e.g.
conventionally in genetic engineering, and may comprise further
genes such as marker genes which allow for the selection of said
vector in a suitable host cell and under suitable conditions.
Furthermore, the vectors may, in addition to the nucleic acid
sequences encoding RGM or its corresponding inhibitiors, comprise
expression control elements, allowing proper expression of the
coding regions in suitable host cells or tissues. Such control
elements are known to the artisan and may include a promoter,
translation initiation codon, translation and insertion site for
introducing an insert into the vector. Preferably, the nucleic acid
molecule of the invention is operatively linked to said expression
control sequences allowing expression in (eukaryotic) cells.
Particularly preferred are in this context control sequences which
allow for correct expression in neuronal cells and/or cells derived
from nervous tissue.
Control elements ensuring expression in eukaryotic cells are well
known to those skilled in the art. As mentioned above, they usually
comprise regulatory sequences ensuring initiation of transcription
and optionally poly-A signals ensuring termination of transcription
and stabilization of the transcript. Additional regulatory elements
may include transcriptional as well as translational enhancers,
and/or naturally-associated or heterologous promoter regions.
Possible regulatory elements permitting expression in for example
mammalian host cells comprise the CMV-HSV thymidine kinase
promoter, SV40, RSV-promoter (Rous sarcoma virus), human elongation
factor 1.alpha.-promoter, CMV enhancer, CaM-kinase promoter or
SV40-enhancer. For the expression for example in nervous tissue
and/or cells derived therefrom, several regulatory sequences are
well known in the art, like the minimal promoter sequence of human
neurofilament L (Charron, J. Biol. Chem 270 (1995), 25739-25745).
Beside elements which are responsible for the initiation of
transcription such regulatory elements may also comprise
transcription termination signals, such as SV40-poly-A site or the
tk-poly-A site, downstream of the polynucleotide. In this context,
suitable expression vectors are known in the art such as
Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pRc/CMV,
pcDNA1, pcDNA3 (In-Vitrogene, as used, inter alia in the appended
examples), pSPORT1 (GIBCO BRL) or pGEMHE (Promega), Beside the
nucleic acid molecules defined herein, the vector may further
comprise nucleic acid sequences encoding for secretion signals.
Such sequences are well known to the person skilled in the art.
Furthermore, depending on the expression system used leader
sequences capable of directing the protein/(poly)peptide to a
cellular compartment may be added to the coding sequence of the
nucleic acid molecules of the invention and are well known in the
art. The leader sequence(s) is (are) assembled in appropriate phase
with translation, initiation and termination sequences, and
preferably, a leader sequence capable of directing secretion of
translated protein, or a part thereof,
As mentioned herein above, said vector may also be, besides an
expression vector, a gene transfer and/or gene targeting vector.
Gene therapy, which is based on introducing therapeutic genes into
cells by ex-vivo or in-vivo techniques is one of the most important
applications of gene transfer. Suitable vectors, vector systems and
methods for in-vitro or in-vivo gene therapy are described in the
literature and are known to the person skilled in the art; see,
e.g., Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ.
Res. 79 (1996), 911-919; Anderson, Science 256 (1992), 808-813,
Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res. 77
(1995), 1077-1086; Wang, Nature Medicine 2 (1996), 714-716; WO
94/29469; WO 97/00957, Schaper, Current Opinion in Biotechnology 7
(1996), 635-640 Verma, Nature 389 (1997), 239-242 WO 94/29469, WO
97/00957, U.S. Pat. No. 5,580,859, U.S. Pat. No. 589,66 or U.S.
Pat. No. 4,394,448 and references cited therein.
In particular, said vectors and/or gene delivery systems are also
described in gene therapy approaches in neurological tissue/cells
(see, inter alia Blomer, J. Virology 71 (1997) 6641-6649) or in the
hypothalamus (see, inter alia, Geddes, Front Neuroendocrinol. 20
(1999), 296-316 or Geddes, Nat. Med. 3 (1997), 1402-1404). Further
suitable gene therapy constructs for use in neurological
cells/tissues are known in the art, for example in Meier (1999), J.
Neuropathol. Exp. Neurol. 58, 1099-1110. The nucleic acid molecules
and vectors of the invention may be designed for direct
introduction or for introduction via liposomes, viral vectors (e.g.
adenoviral, retroviral), electroporation, ballistic (e.g. gene gun)
or other delivery systems into the cell. Additionally, a
baculoviral system can be used as eukaryotic expression system for
the nucleic acid molecules described herein.
The terms "treatment", "treating" and the like are used herein to
generally mean obtaining a desired pharmacological and/or
physiological effect. The effect may be prophylactic in terms of
completely or partially preventing a disease or symptom thereof
and/or may be therapeutic in terms of partially or completely
curing a disease and/or adverse effect attributed to the disease.
The term "treatment" as used herein covers any treatment of a
disease in a mammal, particularly a human, and includes: (a)
preventing the disease from occurring in a subject which may be
predisposed to the disease but has not yet been diagnosed as having
it; (b) inhibiting the disease, i.e. arresting its development; or
(c) relieving the disease, i.e. causing regression of the
disease.
In yet another embodiment, the present invention provides for the
use of a (RGM) polypeptide and/or a polypeptide having or
comprising the amino acid sequence of SEQ ID NOs 18, 20, 23 or 25
or of a functional fragment or derivative thereof or of a
polynucleotide encoding said polypeptide or fragment or derivative
as a marker of stem cells. Since it is envisaged that stem cells as
well as their undifferentiated progenitor cells express RGM, RGM
and/or functional fragments or derivatives thereof may be employed
to influence the differentiation/differentiation pattern of said
stem cells.
It is furthermore envisaged that antibodies directed against RGMs,
in particular directed against polypeptides disclosed herein or
comprising the amino acid sequence of SEQ ID NOs 18, 20, 23 or 25
(or (a) functional fragment(s)/derivative(s) thereof) may be
employed to influence the differentiation of (neuronal) stem cells
and (neuronal) progenitor cells. It is particularly preferred that
said antibodies (as well as other RGM-inhibitors and/or RGM-binding
molecules) be employed to selectively label stem cells. Therefore
these reagents may be employed as markers for stem cells. It is
also envisaged that peptides or derivatives be employed in said
purpose.
In a particularly preferred embodiment of the present invention,
the polypeptide and/or fragment thereof which comprises or has an
amino acid sequence as depicted in SEQ ID NOs 18, 20, 23 or 25
and/or is a RGM molecule to be used in accordance with their
invention is a soluble, i.e. not membrane bound molecule.
As shown in Davis (1994), Science 266, 816-819 ephrins, in
particular A-ephrins, are not active in soluble, monomeric form. In
contrast, soluble RGMs are active and may function without any
membrane-attachment RGM, in contrast to ephrins, is capable of
self-formation of dimers and/or of the formation of higher
aggregates. The invention also provides for the use of a RGM
molecule and/or a polypeptide having or comprising the amino acid
sequence of SEQ ID NOs 18, 20, 23 or 25 or of a functional fragment
or derivative thereof or of a polynucleotide encoding said
polypeptide or a fragment or a derivative for the preparation of a
pharmaceutical composition for alleviating, preventing and/or
treating homeostatic and/or bleeding disorders and/or vascular
damage.
It is envisaged, without being bound by theory, that RGMs may, due
to their structural homology to von-Willebrand factor (vWF), be
employed in the treatment of said disorders/diseases. Furthermore,
it is envisaged that RGM may interact with von-Willebrand factor
and that said molecule, thereby, influences the activity of vWF.
Furthermore, the inhibitors as defined herein should be employed in
disorders where immune cells invade the brain, like multiple
sclerosis, encephalomyelitis disseminata.
The present invention also provides for the use of an antibody or a
fragment or a derivative thereof, or an aptamer, or a binding
molecule capable of interacting with a polypeptide having or
comprising the amino acid sequence of SEQ ID NOs 18, 20, 23 or 25
or with functional fragment or derivative thereof or of an nucleid
acid molecule capable of interacting with a polynucleotide encoding
said polypeptide or a fragment thereof for the preparation of a
diagnostic composition for detecting neurological and/or
neurodegenerative disorders or dispositions thereto.
The diagnostic composition may be used, inter alia, for methods for
determining the expression of the nucleic acids encoding RGM
polypeptides by detecting, inter alia, the presence of the
corresponding mRNA which comprises isolation of RNA from a cell,
contacting the RNA so obtained with a nucleic acid probe as
described above under hybridizing conditions, and detecting the
presence of mRNAs hybridized to the probe.
Furthermore, corresponding mutations and/or alterations may be
detected. Furthermore, RGM (poly)peptides can be detected with
methods known in the art, which comprise, inter alia, immunological
methods, like, ELISA or Western blotting.
The diagnostic composition of the invention may be useful, inter
alia, in detecting the prevalence, the onset or the progress of a
disease related to the aberant expression of a RGM polypeptide.
Accordingly, the diagnostic composition of the invention may be
used, inter alia, for assessing the prevalence, the onset and/or
the disease status of neurological, neurodegenerative and/or
inflammatory disorders, as defined herein above. It is also
contemplated that anti-RGM antibodies, aptamers etc. and
compositions comprising such antibodies, aptamers, etc. may be
useful in discriminating (the) stage(s) of a disease.
The diagnostic composition optionally comprises suitable means for
detection. The nucleic acid molecule(s), vector(s), antibody(ies),
(poly)peptide(s), described above are, for example, suitable for
use in immunoassays in which they can be utilized in liquid phase
or bound to a solid phase carrier. Examples of well-known carriers
include glass, polystyrene, polyvinyl chloride, polypropylene,
polyethylene, polycarbonate, dextran, nylon, amyloses, natural and
modified celluloses, polyacrylamides, agaroses, and magnetite. The
nature of the carrier can be either soluble or insoluble for the
purposes of the invention.
Solid phase carriers are known to those in the art and may comprise
polystyrene beads, latex beads, magnetic beads, colloid metal
particles, glass and/or silicon chips and surfaces, nitrocellulose
strips, membranes, sheets, duracytes and the walls of wells of a
reaction tray, plastic tubes or other test tubes. Suitable methods
of immobilizing nucleic acid molecule(s), vector(s), host(s),
antibody(ies), (poly)peptide(s), fusion protein(s) etc. on solid
phases include but are not limited to ionic, hydrophobic, covalent
interactions and the like. Examples of immunoassays which can
utilize said compounds of the invention are competitive and
non-competitive immunoassays in either a direct or indirect format.
Commonly used detection assays can comprise radioisotopic or
non-radioisotopic methods. Examples of such immunoassays are the
radioimmunoassay (RIA), the sandwich (immunometric assay) and the
Northern or Southern blot assay. Furthermore, these detection
methods comprise, inter alia, IRMA (Immune Radioimmunometric
Assay), EIA (Enzyme Immuno Assay), ELISA (Enzyme Linked Immuno
Assay), FIA (Fluorescent Immuno Assay), and CLIA (Chemioluminescent
Immune Assay). Furthermore, the diagnostic compounds of the present
invention may be are employed in techniques like FRET (Fluorescence
Resonance Energy Transfer) assays.
The nucleic acid sequences encoding RGMs of other species as well
as variants of RGMs are easily deducible from the information
provided herein. These nucleic acid sequences are particularly
useful, as pointed out herein above, in medical and/or diagnostic
setting, but they also provide for important research tools. These
tools may be employed, inter alia, for the generation of transgenic
animals which overexpress or suppress RGMs or wherein the RGM gene
is silenced and/or deleted. Furthermore, said sequences may be
employed to detect and/or ellucidate RGM interaction partners
and/or molecules binding to and/or interfering with RGMs.
THE FIGURES SHOW
FIG. 1: RGM protein fractions Induce collapse of RGC growth
cones.
Solubilized membrane proteins from E9/E10 chick brains were loaded
on two different ion exchange columns, a DEAE anion exchange column
and a cation excange column. RGM was eluted from the cation
exchange column at a NaCl concentration of 200-400 mM in two 1 ml
fractions (4+5) was incorporated into lecithin vesicles and
lecithin vesicles were used in collapse experiments with RGC growth
cones. RGM-containing fractions (4+5, arrows), but not RGM-free
fractions induced extensive collapse (>90%) of RGC growth cones.
Neither ephrin-A5 nor ephrin-A2 could be detected with specific
antibodies, in RGM-fractions. RGC axons and growth cones on laminin
were stained with Alexa-Phalloidin. Western blots from two
dimensional gels were incubated with the F3D4 monoclonal antibody,
and were subsequently stained by a whole protein, india ink
stain.
FIG. 2: Comparative two dimensional gel analysis of tectal proteins
and RGM sequences.
A: Membranes from E9/10 anterior and posterior chick tecta were
enriched and treated with buffer (C) or with PI-PLC (E), to remove
GPI-anchored proteins. The putative RGM (arrow in
Anterior-E+Posterior-E), a PI-PLC cleavable basic protein with a
molecular weight of 33 kDa, was cut out and was used for
nanoelectrospray tandem mass spectrometry. Two dimensional gels
were stained with silver. No anterior-posterior difference of the
RGM candidate is observed in these gels, this is probably due to
the presence of two other proteins in the selected spot. B: Deduced
RGM peptide sequences
FIG. 3: Nucleotide and amino acid sequence of RGM.
A. Nucleotide sequence of RGM.
B. Amino acid sequence of RGM. Peptides derived from
microsequencing are highlighted in bold and peptides used for
making polyclonal antibodies are underlined. Potential
N-glycosylation sites and an RGD tripeptide, potential cell
attachment site are underlined by asterics.
C. Schematic view of the RGM protein. Hydrophobic domains are
present a the N- and C-termini of the protein. Epitopes of the two
polyclonal anti-RGM antibodies are demarcated.
FIG. 4: The polyclonal and the monoclonal RGM antibody recognize
the same 33 kDa protein.
A. The anti-RGM1 antibody binds to a GPI-anchored CRD-(cross
reacting determinant) positive 33 kDa protein. Left blot: An
anti-CRD antibody binds to a low abundant, 33 kDa protein (arrow),
present in the E (PI-PLC supernatant) but not the C fraction
(control supernatant). Right blot: Anti-RGM1 staining of a
GPI-anchored 33 kDa protein on a western blot with supernatant from
E9/E10 chick brain membranes.
B. The GPI-anchored 33 kDa antigen of the anti-RGM1 antibody is
more abundant in posterior (pos.) than in anterior (ant.) tectal
membranes. Left blot: rabbit preimmune serum did not bind to any
protein on western blots with PI-PLC supernatant protein from
anterior and posterior tectum. Right blot: Anti-RGM1 binding to a
33 kDa protein. E=PI-PLC supernatant from tectal membranes,
C=control supernatants from tectal membranes.
C. Anti-RGM1 and F3D4 recognize the same antigens in tectal
membranes. Left blot: F3D4 staining of tectal membrane proteins. A
double band at 33 kDa (lower arrow) and a hardly visible band at 35
kDa (upper arrow) are recognized. Right blot: Anti-RGM1 staining
reveals the same staining pattern with 33 and 35 kDa antigens
(arrows). Contrary to the membrane fraction, where 3 different
protein bands are observed, only one band is detected in most
western blots with PI-PLC supernatants.
For detection on western blots, a secondary, alkaline
phosphatase-conjugated antibody was used and NBT (nitro blue
tetrazolium) and BCIP (bromochloroindolyl phosphat) was used for
the colour reaction.
FIG. 5: The RGM anti-sense probe hybridizes to an mRNA with graded
expression along the anterio-posterior axis.
A, B: RGM-mRNA is expressed in a periventricular gradient in the
tectum of an E9 chick embryo. In a more superficial layer (arrows),
RGM is also expressed but at much lower level. The anterior tectal
pole is to the right, the posterior to the left.
C, D: No staining is detected with the RGM sense probe, on parallel
cryostat sections from E9 chick tecta. The anterior tectal pole is
to the right, the posterior to the left.
FIG. 6: Recombinant RGM induces collapse of retinal growth
cones.
A: RGC axons were grown on laminin-coated coverslips and affinity
purified recombinant RGM was added at a final concentration of 10
ng/ml. More than 90% of temporal retinal growth cones are
collapsed.
B: Neighboring, RGM-free fractions from affinity purification did
not induce collaps of temporal growth cones. Supernatants from
cos-7 cells transfected with an empty plasmid, did not possess any
collapse-inducing activity (data not shown).
In A and B, retinal axons and growth cones were stained with the
F-actin stain Alexa-Phalloidin.
FIG. 7: Recombinant RGM guides temporal retinal axons in the stripe
assay.
A, B: Temporal retinal axons avoid the RGM-containing stripes
(demarcated with red flourescent beads). Membranes from
RGM-transfected cos-7 cells (marked with beads) and anterior tectal
membranes were used to prepare striped carpets.
C, D: Temporal retinal axons do not show any avoidance recation,
when membranes from cos-7 cells, transfected with an empty plasmid
(red beads) were used.
In A-D, striped membrane carpets were in addition coated with
laminin to enhance retinal axon growth in accordance with a
previous protocol (Monschau et al. 1997).
FIG. 8: RGM staining in endothel of (human) brain.
RGM immunoreactivity was detected in endothelial and vascular
smooth muscle cells (SMC), both, in healthy, neuropathological
unaltered control brains and injured brains, suggesting a
constitutive, physiological role in vascular homeostasis.
FIG. 9: RGM expression in a lesion of a human being deceased due to
severe brain injury (1-2 hours after his death). RGM expression on
infiltrating cells from the immune system.
Upregulation of cellular RGM expression correlated with the time
course and appearance of infiltrating leukocytes and activation of
microglia/macrophages after injury (Stoll et al., 1998).
Early after injury (up to 2.5 days), RGM immunoreactivity was found
on leukocytes of granulocytic, monocytic and lymphocytic origin in
vessels within ischemic tissue. Paralleled by edema formation, up
to 1-7 days, RGM-positive cells were found extravasating outside
the vascular walls into the focal ischemic lesioned parenchyma. In
perivascular regions, RGM-positive cells formed clusters in the
Virchow-Robin spaces from day 1-7, which subsided later. These
peri-vascular cells, also referred to as adventitial or perithelial
cells are characteristically alert immune cells (Kato and Walz,
2000; Streit et al., 1999).
FIG. 10: RGM expression in a brain lesion (human).
With increasing time after brain injury, most remarkable changes
corresponded to areas of ongoing scar formation. In these areas,
well defined extracellular RGM-positive laminae and RGM-positive
fibroblastoid and reactive astrocytic cells were visible condensing
adjacent to the border zone. These RGM-positive laminae increased
in magnitude and regional extend over time.
The Examples illustrate the invention.
EXAMPLE I
Microsequencing of an RGM Candidate
To separate RGM from the A-ephrins, a combination of two different
ion exchange columns was employed. RGM, in contrast to the
A-ephrins, bound to a strong cation exchanger and was eluted at a
salt concentration of 200-400 mM NaCl. After incorporation of RGM
into lecithin vesicles, strong collapse-inducing activity was
observed in RGM-fractions (fractions 4+5, FIG. 1) but not in
neighboring RGM-free fractions (fraction 6, FIG. 1). Neither
ephrin-A5 nor ephrin-A2 was present in these fractions, proving
thereby that RGM function does not require presence of the
A-ephrins.
To get peptide sequences from RGM, microsequencing of all proteins,
(cleaved from the membrane by treatment with the enzyme PI-PLC and
having a molecular weight of 30-35 kDa and an isoelectric point
between 7 and 9 was carried out). To this aim, anterior and
posterior membranes from embryonic chick tecta (E9/10) were
prepared with some modifications as described previously (Walter,
Development 101, (1987), 685-96) and membrane pellets were subject
to treatment with enzyme PI-PLC (E fraction) or buffer alone (C
fraction). In particular, Membranes from embryonic chick tecta
(E9/10) were prepared with some modifications as described
previously (Walter et al., 1987). All steps were performed at
4.degree. C. Tecta from 100 chick embryos were isolated and were
divided into three parts equal in length along the
anterior-posterior axis. The middle tectal parts were discarded and
the anterior and posterior parts were worked up separately.
Membranes were washed with PBS containing protease inhibitors and
were centrifuged. Tectal membrane pellets were resuspended in
triethanolamin buffer and were treated with the enzyme PI-PLC (50
mU Boehringer Mannheim/Roche Diagnostics GmbH), to remove
glycosylphosphatidylinositol-anchored (GPI-anchored) proteins from
the membranes. No PI-PLC was added to the other anterior and
posterior tectal membrane fractions, the control-fractions (C).
Enzyme (E) and control (C) fractions were incubated at 37.degree.
C. for 1.5 hours and membrane suspensions were centrifuged at
400.000.times.g in a Beckmann TLA 100.3 rotor. Supernatants were
collected and their protein concentrations were determined
(Bradford 1976, modified by Zor and Selinger, 1996). Supernatants
were precipitated with ice cold 10% trichloroacetic acid, were
centrifuged and protein pellets were washed in ethanol-ether (1:1
v/v) and solubilized in sample buffer (8.5 M urea, 5%
.beta.-mercaptoethanol, 2.5% ampholytes pH 3-10, 2% NP 40).
E fractions and C fractions were loaded onto two dimensional gels,
and after silver staining candidate proteins in the E-fractions
(FIG. 2A, arrows) were cut out and subject to in gel tryptic
digestion and nanoelectrospray ionization (Wilm, Nature 379 (1996),
466-9).
In detail, said 2D gelelectrophoresis and the protein sequence
analysis was carried out as outlined herein below:
Tectal proteins resuspended in sample buffer, were separated using
two dimensional gel electrophoresis. 20 .mu.g of tectal protein was
loaded on each gel. Non-equilibrium pH gradient electrophoresis
(NEPHGE) followed by SDS-PAGE in the second dimension was performed
as described by Boxberg (1988). After the SDS PAGE, gels were
stained by a modified silver staining protocol from Heukeshoven and
Demick (Heukeshoven & Demick, Electrophoresis 9, (1988),
372-375).
Silver-stained proteins in the 2D gels, with a basic isoelectric
point and a molecular weight of 33/35 kDa, present in the PI-PLC
treated E fraction but not in the C fraction, were cut out using a
sharp and sterile scalpel.
Microsequencing was done using the technique of nano-electrospray
tandem mass spectrometry as previously described (Wilm et al.,
1996). The protein spots were digested in gel by trypsin and the
resulting peptides were adsorbed and stepwise eluted into the
electrospray source for mass spectral analysis. Nanoelectrospray
was performed on an API III (Perkin-Elmer) mass spectrometer as
described by Wilm and Mann (Wilm & Mann, Anal. Chem. 68 (1996),
1-8). After selecting an ionized peptide from the peptide mixture,
the peptide was fragmented and the peptide fragments were
analysed.
Mass spectrometric microsequencing of ionized peptides from the
spot marked by arrows in FIG. 2, yielded ten different peptides,
with lengths of 5-14 amino acids as shown in FIG. 2B; (SEQ ID NOs
1-10). The selected spot, was present in anterior and posterior
PI-PLC supernatantes at similar levels. RGM is however more
abundant in posterior than in anterior tectal membranes and the
disappearance of the ap-difference in the 2D-gels was most likely
caused by two different proteins unrelated to RGM and present in
the selected spot.
EXAMPLE II
Cloning of the RGM Gene
Three out of the ten peptide sequences (SEQ ID NOs 1 to 10)
obtained by nanoelectrospray tandem mass spectrometry were used for
synthesis of degenerate oligonucleotide primers and PCR experiments
were performed as follows: Three out of the ten peptide sequences
obtained by nanoelectrospray tandem mass spectrometry were used for
synthesis of degenerate oligonucleotide primers and their
complementary sequences.
TABLE-US-00001 P1F: 5'-ATGCC(AGCT)GA(AG)GA(AG)GT(AGCT)GT(AGCT)-3'
(SEQ ID NO: 11) P1R:
5'-TT(AGCT)AC(AGCT)AC(CT)TC(CT)TC(AGCT)GGCAT-3' (SEQ ID NO: 12)
P2F: 5'-GA(CT)AC(AGCT)TT(CT)CA(AG)AC(AGCT)TG(CT)AA-3' (SEQ ID NO:
13) P2R: 5'-TT(AG)CA(AGCT)GT(CT)TG(AG)AA(AGCT)GT(AG)TC-3' (SEQ ID
NO: 14) P3F: 5'-AA(CT)CA(AG)CA(AG)(CT)T(AGCT)GA(CT)TT(CT)CA-3' (SEQ
ID NO: 15) P3R: 5'-TG(AG)AA(AG)TC(AGCT)A(AG)(CT)TG(CT)TG(AG)TT-3'
(SEQ ID NO: 16)
Moloney murine leukemia virus reverse transcriptase and random
hexamer primers were used to synthesize single-stranded cDNA from
E9 chick tectum total RNA. Combinations of forward (F) and reverse
(R) primers were added to the cDNA and PCR amplification was done
using Taq polymerase. The following PCR conditions were used: an
initial denaturation step at 95.degree. C. for 5 min followed by 30
cycles of 95.degree. C. for 40 s, 50.degree. C. for 1 min,
72.degree. C. for 2 min. The PCR products were cloned into the pGEM
T vector (Promega) and four positive clones were sequenced using
the ALF express sequencer (Pharmacia). The sequence yielded an ORF,
containing most of the peptide sequences obtained by
microsequencing. The 459 bp fragment was used for screening a cDNA
library to obtain the full length sequence and for further analysis
such as Northern bloting and in situ hybridization.
The PCR products were loaded onto agarose gels stained with
ethidium bromide and a PCR product of 459 bp in length, was
obtained and cloned Into the pGEM T vector. After sequencing, most
of the peptide sequences were found in the PCR product, confirming
that the correct candidate was amplified. The 459 bp fragment was
used for screening an E14 chicken brain cDNA library. Positive
clones contained an insert of approximately 4 kb and sequencing
confirmed the presence of the 459 bp fragment and additional
downstream sequences, including a stop codon. Upstream sequences
were obtained by performing 5'-RACE.
In detail, the 459 bp probe was used to screen 500.000 plaques of
an E14 chicken brain library, cloned in the .lamda. Zap vector.
After two screening rounds, eight single plaques were isolated and
the related inserts were cloned into the Bluescript vector using
the rapid excision kit (Stratagene). The positive clones, analysed
by restriction digestions, contained an insert of approximately 4
kb and sequencing confirmed the presence of the 459 bp fragment and
additional downstream sequences, including a stop codon. To get the
sequence of the region upstream of the 459 bp fragment, a 5'-RACE
was performed according to the manufacturer's protocol using the
RACE kit from Boehringer Mannheim and total RNA from E9 chick
tecta. A 700 bp band was amplified, purified, cloned into pGEM T
vector, and 5 positive clones were sequenced. The sequence had an
ORF with two methionines which could act as potential start sites.
The full length sequence of RGM was confirmed independently several
times.
For in situ and Northern blot experiments, the 459 bp fragment was
cloned into the Bluescript KS vector (Stratagene) and anti-sense
and sense probes were produced by using the SP6 and T7 polymerases,
respectively.
This 5'-RACE yielded an ORF with two methionines, potential start
sites. The complete ORF of RGM is 1302 nucleotides in length and
encodes a protein consisting of 434 amino acids (FIG. 3A; SEQ ID
NO:17). Two hydrophobic domains are present at the N-terminus and
C-terminus, respectively (FIG. 3B; SEQ ID NO:18), and two different
algorithms suggested that the N-terminal hydrophobic domain encodes
a signal peptide (best cleavage site predicted: at aa 29), the
C-terminal domain, a GPI-anchor domain (best cleavage site
predicted: at aa 406). RGM has no significant homology to any other
protein, present in the databases and does not carry any specific
domain or motif, except an triamino acid motif, the RGD site, a
potential cell attachment site (Ruoshlahti, Annu. Rev. Cell Dev.
Biol. 12 (1996), 697-715). Preliminary results suggest that this
site is dispensable for RGM function. Polyclonal antibodies, named
anti-RGM1 (against aas: 276-293) and anti-RGM2 (against aas:
110-130), raised against two peptides of the recombinant RGM
molecule, recognize a GPI-anchored 33 kDa molecule, which is
present at higher levels in posterior than in anterior tectal
membrane PI-PLC supernatants (FIG. 4A). In membrane fractions at
least three protein bands appear, a double band at 33 kDa and a
single band at 35 kDa. These protein bands are recognized by the
polyclonal anti-RGM1 antibody and the monoclonal F3D4 antibody
(Muller (1996), loc. cit) (FIG. 4B). Both antibodies show identical
staining patterns on western blots and immunoprecipitation
experiments with anti-RGM1 resulted in pull down of a GPI-anchored,
F3D4-positive protein. These results prove, that the antigens of
the F3D4 monoclonal antibody and of the anti-RGM1 polyclonal
antibody are identical.
RGM is the first member of a new class of axon guidance molecules,
sharing no sequence homology with ephrins, netrins, slits,
semaphorins and any other axon guidance molecules.
The corresponding human RGM sequence (SEQ ID NO:20) could be
deduced by screening the human genome database with the deduced
chicken RGM sequence.
EXAMPLE III
RGM mRNA is Expressed in a Gradient in the Optic Tectum
To analyse expression of RGM-mRNA in the tectum opticum, an RGM
anti-sense probe was used in in situ hybridization experiments on
cryostat sections from E9 chick tecta. Strongest staining is
observed in the periventricular layer, surrounding the tectal
ventricle and staining intensity is much stronger in posterior
tectum than in anterior tectum (FIG. 5A, B). Cell bodies of radial
glial cells are located in the periventricular layer and the
staining pattern confirms previous data using the monoclonal F3D4
antibody, where staining of glial endfeet and of glial cell bodies
was observed (Mueller; (1996), loc. cit.; Mueller, (1997), loc.
cit.). In a more superficial layer, a much weaker staining is
detected with the RGM anti-sense probe but a differential
expression between anterior and posterior tectal poles is hard to
detect in this layer. In this layer tectal neurons are
RGM-positive. This is in line with the expression of RGM by a
subpopulation of tectal neurons. Overall, the staining pattern with
the RGM anti-sense probe looks very similar to the expression
pattern of ephrin-A5 with both messages being found in a
periventricular and in a more superficial tectal layer. No staining
is detectable with the RGM-sense probe.
On northern blots with tectal RNA, the RGM anti-sense probe marked
two transcripts at 5.5 and 6.1 kb. Both messages are down-regulated
at E14 with the smaller message being no longer detectable and the
larger transcript being clearly present, albeit at lower
levels.
RGM is active in in vitro assays and shows a graded expression in
the tectum opticum of vertebrates. Based on Southern blot data it
is assumed that there are least two additional family members which
might have similar guidance activity. (see FIG. 11)
EXAMPLE IV
Recombinant RGM is Active in Collapse and Stripe Assay
To analyse the function of recombinant RGM, the full length RGM
cDNA was used to transfect cos-7 cells with a lipofection
procedure. The full length RGM cDNA was cloned into the KpnI site
of the expression vector pTriEx-1 (Novagen). Cos-7 cells were
transfected with the pTriEx-1 plasmid containing RGM cDNA or with
the empty plasmid using the Superfect transfection reagent (Qiagen)
according to the manufacturer's protocol. The DNA-Superfect mixture
was added to Cos-7 cells growing in 10 cm dishes. 2 hours later
medium was removed, cells were washed with PBS and grown for an
additional 48 hours in fresh medium. Conditioned medium was
collected, run over an RGM-affinity column and RGM-containing
fractions and RGM-free control fractions were directly used in
collapse assay experiments. For stripe assay experiments,
RGM-transfected Cos-7 cells and empty plasmid transfected cells
were washed with PBS and harvested using a rubber policeman in the
presence of homogenization buffer containing protease inhibitors.
Conditioned medium of cos-7 cells transfected with the RGM-pTriEx-1
plasmid was collected and run over an anti-RGM1 antibody column.
Eluted fractions were evaluated with a sensitive and rapid dot blot
assay and RGM-positive fractions were added to retinal axons
growing on a laminin substratum. At a final concentration of 10
ng/ml, soluble RGM induced collapse of 90% of temporal RGC growth
cones (FIG. 6A). Neighboring, RGM-free fractions or conditioned and
concentrated supernatants from cos-7 cells transfected with the
empty plasmid did not possess any collapse-inducing activity (FIG.
6B). Recombinant RGM is active in soluble form, is a strong
difference between RGM and the A-ephrins and suggests a role for a
chemotropic mechanism, in etablishing the retinotectal map.
For preparation of striped membrane carpets, membranes from RGM- or
mock-transfected cells were used. Carpets consisting of alternate
lanes of membranes from mock-transfected cos-7 cells and from
RGM-transfected cells were offered to temporal and nasal RGC axons.
To enhance the poor outgrowth-stimulating activity of cos-7
membranes, anterior tectal membranes or laminin were added.
Collapse assay and stripe assays were prepared and employed as
follows: The collapse assay was performed as descibed (Cox, (1990),
loc. cit.; Wahl, J. Cell Biol. 149(2) (2000), 263-70). 5 .mu.l of
the RGM-positive fraction from the RGM-cos supernatant or
supernatant from control cos cells or RGM-free fractions, was added
to the retinal cultures. One hour later cultures were fixed by
carefully adding 1 ml of fixative (4% paraformaldehyde, 0.33 M
sucrose, pH 7.4). 4-12 hours later, cultures were washed and
stained by Alexa-Phalloidin (Molecular Probes), following the
recommendations of the manufacturer. Stained cultures were stored
on a computer using a CCD camera and the images were analysed with
the SIS analysis imaging software.
Stripe assay experiments were performed as previously described by
Walter et al. (1987). Membrane carpets consisted of lanes of
anterior tectal membranes mixed with membranes from RGM-transfected
cos cells (ratio: 1:1), alternating with lanes consisting of
anterior membranes mixed with membranes from empty plasmid
transfected cos cells (ratio 1:1). In an alternative protocol,
membrane carpets consisting of alternating lanes of membranes from
RGM-transfected cos cells and of control cos membranes, were
incubated for 2 hours at 37.degree. C. with 20 .mu.g/ml laminin
(Becton-Dickinson). Before use, the carpets were washed with Hank's
buffer (2.times.).
On these carpets, temporal RGC axons, but not nasal axons, showed a
clear repulsive avoidance behaviour, growing on the RGM-free
membrane stripes (FIGS. 7A-D). These results demonstrate, that the
recombinant RGM protein is not only active in collapse but also in
stripe assays.
RGM shares with the A-ephrins A2 and A5 the GPI-anchor, the graded
expression and functional activity in two different in vitro
systems. Its activity is however different from the two A-ephrins
in other respects. The specificity of its activity is not
restricted to temporal axons and growth cones. Nasal axons and
growth cones also react, albeit at higher RGM concentrations. This
is in line with the previous observations, that temporal retinal
axons react more strongly to RGM than nasal retinal axons (Stahl,
(1990), loc.cit). For ephrin-A5, a slight difference in sensitivity
of temporal and nasal retinal axons has been observed, this
difference is however not as pronounced as with RGM (Drescher, Cell
82 (1995), 359-70). Besides the stronger concentration dependancy
of RGM function, another crucial difference is that RGM, in
contrast to both ephrin-A5 and ephrin-A2, seems to be active in
soluble form and apparently does not require aggregation to
stimulate its currently unknown retinal receptor. These in vitro
results underscore the difference between ephrins and RGM. In the
stripe assay, inactivation of RGM using the F3D4 monoclonal
antibody and the chromophore-assisted laser inactivation (CALI)
method, resulted in complete neutralization of repulsive guidance
activities of posterior tectal membranes in more than 50% of the
experiments (Mueller, (1996), loc. cit.) F3D4 however neither binds
ephrin-A2 nor ephrin-A5 (Mueller, (1997), loc. cit.) and it was
therefore suggested that the A-ephrins and RGM somehow interact in
special membrane domains to which they are recruited by their
GPI-anchors. Such a colocalization could explain the result, that
inactivation of RGM lead in addition to inactivation of ephrin-A2
and ephrin-A5 and could explain the complete neutralization
observed in the stripe assay experiments (Mueller, (1996), loc.
cit.). The functional relationship of RGM with ephrin-A2 and
ephrin-A5 and the in vivo role of RGM need to be adressed,
especially since both ephrins have been shown to be important
molecular determinants for topographic map formation in vertebrates
(Nakamoto, Cell 86 (1996), 755-66; Frisen, Neuron 20 (1998),
235-43; Feldheim, Neuron 21 (1998), 563-74; Picker, Development 126
(1999), 2967-78; Feldheim, Neuron 25 (2000), 563-74; Brown, Cell
102 (2000), 77-88). There are however evidences from two
vertebrates, which suggest that others factors, besides the
ephrins, are required for formation of the retinotectal map.
Deletion of either the ephrin-A2 or the ephrin-A5 gene in mice,
resulted in mapping phenotypes with some retinal axons forming
ectopic termination zones in the superior colliculus (SC), the
mammalian homologue of the optic tectum, and with nasal retinal
axons overshooting the SC and terminating in the inferior
colliculus. In ephrin-A2.sup.-/- mice, temporal axons showed
mapping errors with ectopic termination zones, but nasal axons did
not show any mapping errors in contrast to the ephrin-A5.sup.-/-
mice which had defects in topographic mapping of nasal but not
temporal axons (Frisen, (1998), loc.cit.; Feldheim, (2000), loc.
cit.). Deletion of both genes should therefore result in a much
more disturbed mapping of both nasal and temporal retinal axons
along the anterior-posterior axis of the SC. This is actually
observed in double mutant ephrin-A2.sup.-/- A5.sup.-/- homozygotes
but a topographic bias of both nasal and temporal axons was still
present, with the majority of temporal and nasal retinal axons
being confined to their anterior and posterior tectal halfs,
respectively (Feldheim, (2000), loc. cit.; Goodhill, Neuron 25
(2000), 501-3). These results point to a role of RGM as one of the
additional factors required for mapping along the
anterior-posterior axis. Such a role is supported by the graded
anterior-low posterior-high expression of this molecule in the SC
of mammals (Mueller, (1997), loc. cit.).
The zebrafish mutant acerebella (ace) is mutant in fgf8 and lacks
the midbrain-hindbrain boundary region and the cerebellum (Reifers,
Development 125 (1998), 2381-95; Picker, (1999), loc. cit.). As a
result the tectum is much smaller in ace mutants than in wildtype
and the expression levels of all three zebrafish A-ephrins are
changed with ephrin-A2 and ephrin-A5a being still expressed at low
and anterior levels in ace tecta and with ephrin-A5b being
completely eliminated (Picker et al., 1999). In ace mutant tecta,
mapping of retinal axons along the anterior-posterior axis is
normal in dorsal tectum and is not completely lost in ventral
tectum, suggesting the involvement of other graded guidance cues,
not seriously affected by the fgf8 mutation in the ace zebrafish
mutants (Picker et al., 1999). Dorsoventral patterning in both
zebrafish ace mutants and ephrin-A2.sup.-/- A5.sup.-/- double knock
out mice is affected.
RGM, with its graded expression along the anterior-posterior axis
of the tectum and its ability to function in a secreted and
membrane-coupled way, is an important player for topographic map
formation.
EXAMPLE V
Materials and Methods
1. Patients
21 brains of patients with clinical history and neuropathologically
confirmed diagnosis of focal cerebral infarctions and 25 brains of
patients with traumatic brain injury were included in this study.
Infarctioned brain tissue was derived from an updated stroke and
trauma brain-bank (Table 1,2) reported previously (Postler et al.,
1997, Beschorner et al., 2000). Tissue specimen procurement was
performed according to the ethical guidelines of the University of
Tuebingen. Patients with altered immune status because of
immunosuppressive therapy or meningitis/encephalitis were excluded
from this study. As controls, the results were compared to tissue
from corresponding areas of 4 normal non-ischemic brains described
previously (Schwab et al., 2000). In addition to patient data,
haematoxyline-eosine (HE), luxol fast blue (LFB) and iron (Fe)
staining was used for evaluation of the typical histological
features defined as standard indication of infarct (Kalimo et al.,
1996) and trauma age (Graham and Gennarelli, 1996).
2. Immunohistochemistry
After formaldehyde fixation and paraffin-embedding, rehydrated 2
.mu.m sections were boiled (in an 600 W microwave oven) seven times
for 5 min in citrate buffer (2.1 g sodium citrate/liter, pH 7.4).
Endogenous peroxsidase was inhibited with 1% H.sub.2O.sub.2 in
methanol (1:10; 15 min). Sections were incubated with 10% normal
porcine serum (Biochrom, Berlin, FRG) to block non-specific binding
of immunoglobulins. Monospecific polyclonal antibodies directed
against RGM were diluted (1:10) in 1% BSA (bovine serum albumin)
TBS (Tris-balanced salt solution, containing 0.025 M Tris, 0.15 M
NaCl) and incubated over night at room temperature. Specific
binding of the antibodies were detected with a secondary
biotinylated swine anti-rabbit IgG F(ab).sub.2 antibody fragment
1:400 for 30 min (DAKO, Hamburg, FRG), followed by incubation with
a peroxidase conjugated streptavidin-biotin complex (DAKO, Hamburg,
FRG). The enzyme was visualized with diaminobenzidine as a
chromogen (Fluka, Neu-Ulm, FRG). Sections were counterstained with
Mayer's Hemalaun. Negative controls consisted of sections incubated
in the absence of the primary antibody. Specificity of polyclonal
RGM antibody was confirmed by inhibition of staining using human
ischemic brain tissue after pre-incubation for 3 h on ice with
access of the cognate RGM peptide.
3. Double Labeling Experiments
In double labelling experiments, a cell-type or activation specific
antigen was first labelled using the ABC procedure in combination
with alkaline phosphatase conjugates. Specific antigens were
labelled with antibodies against GFAP (glial fibrillary acidic
protein, monoclonal, Boehringer Mannheim, Germany, 1:100) to detect
astrocytes, MBP (myelin basic protein, polyclonal,
oligodendrocytes, Dako, 1:500) and CD68 (Dako, 1:100) for
microglia/macrophage identification. Activated
microglia/macrophages were detected with antibodies directed
against HLA-DR, -DP, -DQ (MHC class II, DAKO, Glostrup, Denmark,
1:100) or MRP-8 (8-5C2, BMA, Augst, Switzerland, 1:100) (Postler et
al., 1997). Lymphocytic subpopulations were classified with
monoclonal antibodies against CD4 (T-helper lymphocytes, 1:10,
Dako) and CD8 (T cytotoxic/suppressor lymphocytes, 1:500, Dako) and
CD20 (pan B cell marker, 1:200, Dako). In order to detect
extracellular basal lamina structures in vessels and during scar
formation mouse laminin (1:500, Chemicon) antibodies were used and
rabbit fibronectin (1:100, Dako) antibodies were used to detect
matrix deposition. Furthermore, in order to characterize the
cellular proliferation response, sections were incubated with the S
phase specific PCNA (proliferating cell nuclear antigen, 1:100,
Dako) monoclonal antibodies. Briefly, slices were deparaffinized,
irradiated in a microwave oven for antigen retrieval and incubated
with non specific porcine serum as described above. Visualization
was achieved by adding biotinylated secondary antibodies (1:400)
for 30 min and alkaline phosphatase conjugated ABC complex diluted
1:400 in TBS-BSA for 30 min. Consecutively, slices were developed
with Fast-Blue BB salt chromogen-substrate solution yielding a blue
reaction product. Between double labelling experiments, slices were
irradiated in a microwave for 5 min in citrate buffer. Then RGM was
immunodetected as described above.
4. Evaluation and Statistical Analysis
Data were calculated as means of labelled cells (MLC, .+-.SEM) from
border zones or remote areas of the same tissue section and were
compared to normal control brains using the two-tailed unpaired
student's t-test. Border zones were defined as peri-lesional areas
adjacent to the developing necrotic core demarcating the region of
major damage. RGM.sup.+ cells were counted in ten high power fields
(HPF, .times.200 magnification with an eye-piece-grid representing
0.25 mm.sup.2).
Results
21 brains of patients with focal cerebral infarctions (FCI), 25
brains with traumatic brain injury (TBI) and 4 control brains were
evaluated for RGM protein expression by immunohistochemistry.
1. Healthy, Neuropathological Unaltered Control Brains
In control brains without neuropathologically alterations, RGM
immunoreactivity was detected on white matter fibres,
oliogodendrocytes, the perikarya of some neurons and RGM.sup.+
cells were also detected in the choroid plexus (FIG. 8) and
ependyma. Only single cells were detected in peri-vascular spaces.
Further, some smooth muscle cells and few endothelial cells but no
astrocytes were labelled.
2. Focal Cerebral Ischemia (FCI)
It was analysed whether number and distribution of RGM expressing
cells is altered after cerebral infarctions. Results suggested,
that RGM expression is lesion-associated. Cellular RGM expression
was confined to neurons, few reactive astrocytes and invading
leukocytes. With the ageing of the lesions, RGM-positive
extracellular laminae components were found in the constituting
scar.
RGM-positive cells accumulated in infarctioned white matter,
hemorrhagic areas, infarction core and peri-infarctional areas,
respectively. Using the students t-test, a significantly
(P<0.0001) higher number of RGM.sup.+ cells was detected in
peri-infarcional areas (MLC=24, SEM=1.1) than in remote areas
(MLC=2, SEM=0.2) or control tissue (MLC=6, SEM=0.8). The
morphological described peri-infarctional areas were part of the
physiologically defined penumbra. In these areas the number of
RGM-positive cells accumulated already up to day 1 (p<0.0001,
MLC=31.93, SEM=2.3) reached their maximum 1.5-2.5 days (MLC=34,
SEM=3.2) after infarction and remained elevated up to several weeks
and months of survival (MLC=11, SEM=1.4). Early after ischemic
damage (up to 2.5 days), RGM immunoreactivity was predominantly
found on neurons and leukocytes of granulocytic, monocytic and
lymphocytic origin in vessels within ischemic tissue. Paralleled by
edema formation, up to 1-7 days, RGM-positive cells were found
extravasating outside the vascular walls into the focal ischemic
lesioned parenchyma. In perivascular regions, RGM-positive cells
formed clusters in the Virchow-Robin spaces from day 1-7, which
subsided later. These peri-vascular cells, also referred to as
adventitial or perithelial cells are characteristically alert
immune cells (Kato and Walz, 2000; Streit et al., 1999). With
lesion aging, from day 3 onwards, lesional RGM expression by few
reactive astrocytes, was observed. At later stages, arising 1 week
after infarction, extracellular RGM deposits were detected
constituting neo-laminae localized to areas of ongoing scar
formation. These RGM-positive laminae increased in magnitude and
regional extend over time. With tissue reorganisation of the
lesion, also "foamy", lipid loaded RGM-positive phagocytic
RGM-positive microglia/macrophages were observed.
Upregulation of cellular RGM expression correlated with the time
course and appearance of infiltrating leukocytes and activation of
microglia/macrophages after injury (Stoll et al., 1998). Whereas
upregulation of extracellular RGM expression correlated with the
time course and the appearance of the scar after injury. In few
cases (<5% of counterstained nuclei) some reactive astrocytes
restricted to the demarcating lesion core also expressed RGM.
3. Traumatic Brain Injury
In patients who died after TBI, in accordance to cerebral
infarction (FCI) the immunohistological evaluation revealed early
cellular membranous, cytoplasmatic and nuclear RGM expression by
leukocytes, few reactive astrocytes and neurons with strong
staining of their perikarya, dendrites and axons (FIG. 9). During
the observed time post TBI, within the necrotic core and the
bordering peri-necrotic parenchyma accumulation of RGM-positive
cells (p<0.0001) was detected in border zones (MLC=22, SEM=0.7)
compared to remote areas (MLC=1, SEM=0.1) and normal brain controls
(MLC=5.8, SEM=0.8). Following TBI, RGM-positive cell numbers arose
already during the first 24 hours (p<0.0001) where RGM-positive
cell numbers reached maximum levels (MLC=29, SEM=0.9) and decreased
subsequently. With increasing time after TBI, most remarkable
changes corresponded to areas of ongoing scar formation (FIG. 10).
In these areas, well defined extracellular RGM-positive laminae
were visible condensing adjacent to the border zone. RGM
immunoreactivity was also detected in endothelial and vascular
smooth muscle cells (SMC) but no significant differences were
observed between injured and control brains.
References as mentioned in the example herein above: Beschomer,
Acta Neuropathol. 100 (2000), 377-384 Graham, "Greenfield's
Neuropathology." D. I. Graham and P. L. Lantos (eds), 6.sup.th.
Edn., Edward Arnold, London (1996), pps. 197-248 Kalimo,
Greenfield's Neuropathology 6.sup.th. Edn. Arnold, London Sydney
Auckland (1996), pp 315-381. Kato, Brain Pathol., 10 (2000),
137-143. Postler, Glia 19 (1997), 27-34. Schwab, Acta Neuropathol.
99 (2000), 609-614. Stoll, Prog. Neurobiol. 56 (1998), 149-171.
Streit, Prog. Neurobiol. 57 (1999), 563-581.
EXAMPLE VI
Change of Tumor Growth Behaviour in Mice
Hybridoma cells secreting the RGM-specific F3D4 monoclonal antibody
were injected into the peritoneum of mice, primed with mineral oil.
Normally the hybridoma cells continue to divide in the peritoneum
and the hybridoma cells secreted large amounts of antibody,
resulting in formation of large ascites tumors. Mice receiving the
F3D4-producing hybridoma cells did not develop ascites tumors in
the peritoneal cavity, but developed solid, adherent tumors. The
F3D4 monoclonal antibody resulted in a change of phenotype of the
tumorigenic hybridoma cells from a less invasive, non-adherent
state to an invasive adherent state. Masking of endogeneous RGM by
the antibodies secreted from the hybridoma cells, enabled adhesion
and invasion of these tumor cells and was responsible for this
outcome.
EXAMPLE VII
Detection of a Functional RGM Fragment
RGM was cloned into the pTriEx vector and the vector was cut inside
the polylinker side and inside the RGM sequence using SacI in the
first step. After ligation of both ends the RGM containing vector
was cut in the second step with StuI inside the RGM sequence and in
the polylinker with PmII. After ligation of both ends, the vector
with the shorter RGM-fragments was transfected into COS7 cells.
Cell lysates of these COS cells were purified using an anti-RGM1
affinity column and RGM-containing fractions were used in collapse
assay experiments. A fragment as described in SEQ ID NO:19 was
active in said assays.
EXAMPLE VIII
Detection of Further RGMs
A publicly available computer database at the National Center for
Biotechnology Information (NCBI, USA) was used to identify human
genes homologous to chicken RGM, employing the information and data
illustrated in the examples herein above. A search strategy based
on the Blast algorithm (NCBI) resulted in three human genes located
on chromosomes 1, 5 and 15. The corresponding contigs are
NT.sub.--021932.5 (RGM3), NT.sub.--029283.2 (RGM2) and
NT.sub.--010370.5 (RGM1), respectively. cDNA sequences for RGM 1, 2
and 3 were derived from these genomic sequences by omitting introns
and fusing the remaining exons.
Corresponding aminio acid and nucleotide sequences for human RGM2
are illustrated in appended SEQ ID NOs: 22 and 23. Human
RGM3-sequences are shown in SEQ ID NOs: 24 and 25.
SEQUENCE LISTINGS
1
2519PRTArtificial SequenceDescription of Artificial Sequence
synthetic 1Tyr Leu Gly Thr Thr Leu Val Val Arg 1 527PRTArtificial
SequenceDescription of Artificial Sequence synthetic 2Thr Phe Thr
Asp Thr Phe Gln 1 5312PRTArtificial SequenceDescription of
Artificial Sequence synthetic 3Met Pro Glu Glu Val Val Asn Ala Val
Glu Asp Arg 1 5 1046PRTArtificial SequenceDescription of Artificial
Sequence synthetic 4Leu Thr Leu Leu Phe Lys 1 5510PRTArtificial
SequenceDescription of Artificial Sequence synthetic 5Thr Phe Thr
Asp Thr Phe Gln Thr Cys Lys 1 5 10614PRTArtificial
SequenceDescription of Artificial Sequence synthetic 6Gly Cys Pro
Leu Asn Gln Gln Leu Asp Phe Gln Thr Met Arg 1 5 1075PRTArtificial
SequenceDescription of Artificial Sequence synthetic 7Ala Glu Met
Asp Glu 1 587PRTArtificial SequenceDescription of Artificial
Sequence synthetic 8Pro Glu Ala Phe Thr Tyr Glu 1 595PRTArtificial
SequenceDescription of Artificial Sequence synthetic 9His Leu Glu
Tyr Arg 1 5105PRTArtificial SequenceDescription of Artificial
Sequence synthetic 10Gln Gly Leu Tyr Leu 1 51129DNAArtificial
SequenceDescription of Artificial Sequence synthetic 11atgccagctg
aaggaaggta gctgtagct 291231DNAArtificial SequenceDescription of
Artificial Sequence synthetic 12ttagctacag ctaccttcct tcagctggca t
311330DNAArtificial SequenceDescription of Artificial Sequence
synthetic 13gactacagct ttctcaagac agcttgctaa 301430DNAArtificial
SequenceDescription of Artificial Sequence synthetic 14ttagcaagct
gtcttgagaa agctgtagtc 301529DNAArtificial SequenceDescription of
Artificial Sequence synthetic 15aactcaagca agcttagctg actttctca
291629DNAArtificial SequenceDescription of Artificial Sequence
synthetic 16tgagaaagtc agctaagctt gcttgagtt 29171302DNAArtificial
SequenceDescription of Artificial Sequence synthetic 17atgggtatgg
ggagaggggc aggatccaca gccctgggac ttttccaaat cctccctgtc 60tttctctgca
tcttccctcc agtgacgtct ccatgcaaga tcctcaagtg caactctgag
120ttctgggcgg ccacgtcggg ttcgcaccac ctgggcgcag aggaaacccc
ggagttctgc 180acggcgttgc gcgcctacgc gcactgcacc cgccgcaccg
cccgcacctg caggggggac 240ctggcctacc actcggccgt gcatggcata
gacgatctca tggtgcaaca caactgctcc 300aaggatggcc ccacgtccca
gccccgcctc cggacattgc cccccgggga cagccaggag 360cgctctgaca
gccccgaaat ctgccactac gagaagagct ttcacaaaca ctcggcagct
420cccaactaca cccactgtgg gctcttcggg gacccccacc tcaggacttt
cacggacacc 480ttccagacct gcaaggtgca aggggcttgg ccgctcatag
acaataacta cctgaacgtc 540caggtcacca acacgccggt gctgcctggc
tcctcagcca ccgccaccag caagctcacc 600atcatcttca agagcttcca
ggaatgcgtg gagcagaaag tgtaccaggc agagatggac 660gagctccctg
ctgcctttgc tgatggctcc aagaacggcg gcgacaagca cggagccaac
720agcctgaaga tcaccgagaa ggtgtcgggc cagcacatcg agatccaggc
caagtacatt 780ggcaccacca tcgtggtgag gcaggtgggc cgctacctca
ccttcgccgt gcgtatgccg 840gaggaggtgg tcaacgctgt ggaggaccgg
gacagtcagg gcctctacct gtgcctccgg 900ggttgtccgc tcaaccaaca
gattgacttc cagactttcc gcttggctca ggccgctgag 960ggccgtgctc
gcaggaaggg gcccagcttg ccggcccccc ctgaggcctt cacttacgag
1020tcggccactg ccaagtgcag ggaaaagctg cccgtagagg acctctactt
ccagtcctgc 1080gtctttgacc tcctgactac gggggatgtc aacttcatgc
tggctgctta ttacgctttt 1140gaggacgtga agatgcttca ctccaacaaa
gacaaactgc acctctatga aaggacacgg 1200gccctagccc cgggcaatgc
agctccctcg gagcatccct gggccctccc tgccctctgg 1260gtagcactgc
tgagtttgag tcagtgttgg ttgggtttgt ta 130218434PRTGallus gallus 18Met
Gly Met Gly Arg Gly Ala Gly Ser Thr Ala Leu Gly Leu Phe Gln 1 5 10
15Ile Leu Pro Val Phe Leu Cys Ile Phe Pro Pro Val Thr Ser Pro Cys
20 25 30Lys Ile Leu Lys Cys Asn Ser Glu Phe Trp Ala Ala Thr Ser Gly
Ser 35 40 45His His Leu Gly Ala Glu Glu Thr Pro Glu Phe Cys Thr Ala
Leu Arg 50 55 60Ala Tyr Ala His Cys Thr Arg Arg Thr Ala Arg Thr Cys
Arg Gly Asp 65 70 75 80Leu Ala Tyr His Ser Ala Val His Gly Ile Asp
Asp Leu Met Val Gln 85 90 95His Asn Cys Ser Lys Asp Gly Pro Thr Ser
Gln Pro Arg Leu Arg Thr 100 105 110Leu Pro Pro Gly Asp Ser Gln Glu
Arg Ser Asp Ser Pro Glu Ile Cys 115 120 125His Tyr Glu Lys Ser Phe
His Lys His Ser Ala Ala Pro Asn Tyr Thr 130 135 140His Cys Gly Leu
Phe Gly Asp Pro His Leu Arg Thr Phe Thr Asp Thr145 150 155 160Phe
Gln Thr Cys Lys Val Gln Gly Ala Trp Pro Leu Ile Asp Asn Asn 165 170
175Tyr Leu Asn Val Gln Val Thr Asn Thr Pro Val Leu Pro Gly Ser Ser
180 185 190Ala Thr Ala Thr Ser Lys Leu Thr Ile Ile Phe Lys Ser Phe
Gln Glu 195 200 205Cys Val Glu Gln Lys Val Tyr Gln Ala Glu Met Asp
Glu Leu Pro Ala 210 215 220Ala Phe Ala Asp Gly Ser Lys Asn Gly Gly
Asp Lys His Gly Ala Asn225 230 235 240Ser Leu Lys Ile Thr Glu Lys
Val Ser Gly Gln His Ile Glu Ile Gln 245 250 255Ala Lys Tyr Ile Gly
Thr Thr Ile Val Val Arg Gln Val Gly Arg Tyr 260 265 270Leu Thr Phe
Ala Val Arg Met Pro Glu Glu Val Val Asn Ala Val Glu 275 280 285Asp
Arg Asp Ser Gln Gly Leu Tyr Leu Cys Leu Arg Gly Cys Pro Leu 290 295
300Asn Gln Gln Ile Asp Phe Gln Thr Phe Arg Leu Ala Gln Ala Ala
Glu305 310 315 320Gly Arg Ala Arg Arg Lys Gly Pro Ser Leu Pro Ala
Pro Pro Glu Ala 325 330 335Phe Thr Tyr Glu Ser Ala Thr Ala Lys Cys
Arg Glu Lys Leu Pro Val 340 345 350Glu Asp Leu Tyr Phe Gln Ser Cys
Val Phe Asp Leu Leu Thr Thr Gly 355 360 365Asp Val Asn Phe Met Leu
Ala Ala Tyr Tyr Ala Phe Glu Asp Val Lys 370 375 380Met Leu His Ser
Asn Lys Asp Lys Leu His Leu Tyr Glu Arg Thr Arg385 390 395 400Ala
Leu Ala Pro Gly Asn Ala Ala Pro Ser Glu His Pro Trp Ala Leu 405 410
415Pro Ala Leu Trp Val Ala Leu Leu Ser Leu Ser Gln Cys Trp Leu Gly
420 425 430Leu Leu19116PRTGallus gallus 19Glu Leu Pro Ala Ala Phe
Ala Asp Gly Ser Lys Asn Gly Gly Asp Lys 1 5 10 15His Gly Ala Asn
Ser Leu Lys Ile Thr Glu Lys Val Ser Gly Gln His 20 25 30Ile Glu Ile
Gln Ala Lys Tyr Ile Gly Thr Thr Ile Val Val Arg Gln 35 40 45Val Gly
Arg Tyr Leu Thr Phe Ala Val Arg Met Pro Glu Glu Val Val 50 55 60Asn
Ala Val Glu Asp Arg Asp Ser Gln Gly Leu Tyr Leu Cys Leu Arg 65 70
75 80Gly Cys Pro Leu Asn Gln Gln Ile Asp Phe Gln Thr Phe Arg Leu
Ala 85 90 95Gln Ala Ala Glu Gly Arg Ala Arg Arg Lys Gly Pro Ser Leu
Pro Ala 100 105 110Pro Pro Glu Ala 11520434PRTHomo sapiens 20Met
Gly Met Gly Arg Gly Ala Gly Arg Ser Ala Leu Gly Phe Trp Pro 1 5 10
15Thr Leu Ala Phe Leu Leu Cys Ser Phe Pro Ala Ala Thr Ser Pro Cys
20 25 30Lys Ile Leu Lys Cys Asn Ser Glu Phe Trp Ser Ala Thr Ser Gly
Ser 35 40 45His Ala Pro Ala Ser Asp Asp Thr Pro Glu Phe Cys Ala Ala
Leu Arg 50 55 60Ser Tyr Ala Leu Cys Thr Arg Arg Thr Ala Arg Thr Cys
Arg Gly Asp 65 70 75 80Leu Ala Tyr His Ser Ala Val His Gly Ile Glu
Asp Leu Met Ser Gln 85 90 95His Asn Cys Ser Lys Asp Gly Pro Thr Ser
Gln Pro Arg Leu Arg Thr 100 105 110Leu Pro Pro Ala Gly Asp Ser Gln
Glu Arg Ser Asp Ser Pro Glu Ile 115 120 125Cys His Tyr Glu Lys Ser
Phe His Lys His Ser Ala Thr Pro Asn Tyr 130 135 140Thr His Cys Gly
Leu Phe Gly Asp Pro His Leu Arg Thr Phe Thr Asp145 150 155 160Arg
Phe Gln Thr Cys Lys Val Gln Gly Ala Trp Pro Leu Ile Asp Asn 165 170
175Asn Tyr Leu Asn Val Gln Val Thr Asn Thr Pro Val Leu Pro Gly Ser
180 185 190Ala Ala Thr Ala Thr Ser Lys Leu Thr Ile Ile Phe Lys Asn
Phe Gln 195 200 205Glu Cys Val Asp Gln Lys Val Tyr Gln Ala Glu Met
Asp Glu Leu Pro 210 215 220Ala Ala Phe Val Asp Gly Ser Lys Asn Gly
Gly Asp Lys His Gly Ala225 230 235 240Asn Ser Leu Lys Ile Thr Glu
Lys Val Ser Gly Gln His Val Glu Ile 245 250 255Gln Ala Lys Tyr Ile
Gly Thr Thr Ile Val Val Arg Gln Val Gly Arg 260 265 270Tyr Leu Thr
Phe Ala Val Arg Met Pro Glu Glu Val Val Asn Ala Val 275 280 285Glu
Asp Trp Asp Ser Gln Gly Leu Tyr Leu Cys Leu Arg Gly Cys Pro 290 295
300Leu Asn Gln Gln Ile Asp Phe Gln Ala Phe His Thr Asn Ala Glu
Gly305 310 315 320Thr Gly Ala Arg Arg Leu Ala Ala Ala Ser Pro Ala
Pro Thr Ala Pro 325 330 335Glu Thr Phe Pro Tyr Glu Thr Ala Val Ala
Lys Cys Lys Glu Lys Leu 340 345 350Pro Val Glu Asp Leu Tyr Tyr Gln
Ala Cys Val Phe Asp Leu Leu Thr 355 360 365Thr Gly Asp Val Asn Phe
Thr Leu Ala Ala Tyr Tyr Ala Leu Glu Asp 370 375 380Val Lys Met Leu
His Ser Asn Lys Asp Lys Leu His Leu Tyr Glu Arg385 390 395 400Thr
Arg Asp Leu Pro Gly Arg Ala Ala Ala Gly Leu Pro Leu Ala Pro 405 410
415Arg Pro Leu Leu Gly Ala Leu Val Pro Leu Leu Ala Leu Leu Pro Val
420 425 430Phe Cys211305DNAHomo sapiens 21atgggtatgg ggagaggggc
aggacgttca gccctgggat tctggccgac cctcgccttc 60cttctctgca gcttccccgc
agccacctcc ccgtgcaaga tcctcaagtg caactctgag 120ttctggagcg
ccacgtcggg cagccacgcc ccagcctcag acgacacccc cgagttctgt
180gcagccttgc gcagctacgc cctgtgcacg cggcggacgg cccgcacctg
ccggggtgac 240ctggcctacc actcggccgt ccatggcata gaggacctca
tgagccagca caactgctcc 300aaggatggcc ccacctcgca gccacgcctg
cgcacgctcc caccggccgg agacagccag 360gagcgctcgg acagccccga
gatctgccat tacgagaaga gctttcacaa gcactcggcc 420acccccaact
acacgcactg tggcctcttc ggggacccac acctcaggac tttcaccgac
480cgcttccaga cctgcaaggt gcagggcgcc tggccgctca tcgacaataa
ttacctgaac 540gtgcaggtca ccaacacgcc tgtgctgccc ggctcagcgg
ccactgccac cagcaagctc 600accatcatct tcaagaactt ccaggagtgt
gtggaccaga aggtgtacca ggctgagatg 660gacgagctcc cggccgcctt
cgtggatggc tctaagaacg gtggggacaa gcacggggcc 720aacagcctga
agatcactga gaaggtgtca ggccagcacg tggagatcca ggccaagtac
780atcggcacca ccatcgtggt gcgccaggtg ggccgctacc tgacctttgc
cgtccgcatg 840ccagaggaag tggtcaatgc tgtggaggac tgggacagcc
agggtctcta cctctgcctg 900cggggctgcc ccctcaacca gcagatcgac
ttccaggcct tccacaccaa tgctgagggc 960accggtgccc gcaggctggc
agccgccagc cctgcaccca cagcccccga gaccttccca 1020tacgagacag
ccgtggccaa gtgcaaggag aagctgccgg tggaggacct gtactaccag
1080gcctgcgtct tcgacctcct caccacgggc gacgtgaact tcacactggc
cgcctactac 1140gcgttggagg atgtcaagat gctccactcc aacaaagaca
aactgcacct gtatgagagg 1200actcgggacc tgccaggcag ggcggctgcg
gggctgcccc tggccccccg gcccctcctg 1260ggcgccctcg tcccgctcct
ggccctgctc cctgtgttct gctag 1305221314DNAHomo sapiens 22atgggcttga
gagcagcacc ttccagcgcc gccgctgccg ccgccgaggt tgagcagcgc 60cgcagccccg
ggctctgccc cccgccgctg gagctgctgc tgctgctgct gttcagcctc
120gggctgctcc acgcaggtga ctgccaacag ccagcccaat gtcgaatcca
gaaatgcacc 180acggacttcg tgtccctgac ttctcacctg aactctgccg
ttgacggctt tgactctgag 240ttttgcaagg ccttgcgtgc ctatgctggc
tgcacccagc gaacttcaaa agcctgccgt 300ggcaacctgg tataccattc
tgccgtgttg ggtatcagtg acctcatgag ccagaggaat 360tgttccaagg
atggacccac atcctctacc aaccccgaag tgacccatga tccttgcaac
420tatcacagcc acgctggagc cagggaacac aggagagggg accagaaccc
tcccagttac 480cttttttgtg gcttgtttgg agatcctcac ctcagaactt
tcaaggataa cttccaaaca 540tgcaaagtag aaggggcctg gccactcata
gataataatt atctttcagt tcaagtgaca 600aacgtacctg tggtccctgg
atccagtgct actgctacaa ataagatcac tattatcttc 660aaagcccacc
atgagtgtac agatcagaaa gtctaccaag ctgtgacaga tgacctgccg
720gccgcctttg tggatggcac caccagtggt ggggacagcg atgccaagag
cctgcgtatc 780gtggaaaggg agagtggcca ctatgtggag atgcacgccc
gctatatagg gaccacagtg 840tttgtgcggc aggtgggtcg ctacctgacc
cttgccatcc gtatgcctga agacctggcc 900atgtcctacg aggagagcca
ggacctgcag ctgtgcgtga acggctgccc cctgagtgaa 960cgcatcgatg
acgggcaggg ccaggtgtct gccatcctgg gacacagcct gcctcgcacc
1020tccttggtgc aggcctggcc tggctacaca ctggagactg ccaacactca
atgccatgag 1080aagatgccag tgaaggacat ctatttccag tcctgtgtct
tcgacctgct caccactggt 1140gatgccaact ttactgccgc agcccacagt
gccttggagg atgtggaggc cctgcaccca 1200aggaaggaac gctggcacat
tttccccagc agtggcaatg ggactccccg tggaggcagt 1260gatttgtctg
tcagtctagg actcacctgc ttgatcctta tcgtgttttt gtag 131423437PRTHomo
sapiens 23Met Gly Leu Arg Ala Ala Pro Ser Ser Ala Ala Ala Ala Ala
Ala Glu 1 5 10 15Val Glu Gln Arg Arg Ser Pro Gly Leu Cys Pro Pro
Pro Leu Glu Leu 20 25 30Leu Leu Leu Leu Leu Phe Ser Leu Gly Leu Leu
His Ala Gly Asp Cys 35 40 45Gln Gln Pro Ala Gln Cys Arg Ile Gln Lys
Cys Thr Thr Asp Phe Val 50 55 60Ser Leu Thr Ser His Leu Asn Ser Ala
Val Asp Gly Phe Asp Ser Glu 65 70 75 80Phe Cys Lys Ala Leu Arg Ala
Tyr Ala Gly Cys Thr Gln Arg Thr Ser 85 90 95Lys Ala Cys Arg Gly Asn
Leu Val Tyr His Ser Ala Val Leu Gly Ile 100 105 110Ser Asp Leu Met
Ser Gln Arg Asn Cys Ser Lys Asp Gly Pro Thr Ser 115 120 125Ser Thr
Asn Pro Glu Val Thr His Asp Pro Cys Asn Tyr His Ser His 130 135
140Ala Gly Ala Arg Glu His Arg Arg Gly Asp Gln Asn Pro Pro Ser
Tyr145 150 155 160Leu Phe Cys Gly Leu Phe Gly Asp Pro His Leu Arg
Thr Phe Lys Asp 165 170 175Asn Phe Gln Thr Cys Lys Val Glu Gly Ala
Trp Pro Leu Ile Asp Asn 180 185 190Asn Tyr Leu Ser Val Gln Val Thr
Asn Val Pro Val Val Pro Gly Ser 195 200 205Ser Ala Thr Ala Thr Asn
Lys Ile Thr Ile Ile Phe Lys Ala His His 210 215 220Glu Cys Thr Asp
Gln Lys Val Tyr Gln Ala Val Thr Asp Asp Leu Pro225 230 235 240Ala
Ala Phe Val Asp Gly Thr Thr Ser Gly Gly Asp Ser Asp Ala Lys 245 250
255Ser Leu Arg Ile Val Glu Arg Glu Ser Gly His Tyr Val Glu Met His
260 265 270Ala Arg Tyr Ile Gly Thr Thr Val Phe Val Arg Gln Val Gly
Arg Tyr 275 280 285Leu Thr Leu Ala Ile Arg Met Pro Glu Asp Leu Ala
Met Ser Tyr Glu 290 295 300Glu Ser Gln Asp Leu Gln Leu Cys Val Asn
Gly Cys Pro Leu Ser Glu305 310 315 320Arg Ile Asp Asp Gly Gln Gly
Gln Val Ser Ala Ile Leu Gly His Ser 325 330 335Leu Pro Arg Thr Ser
Leu Val Gln Ala Trp Pro Gly Tyr Thr Leu Glu 340 345 350Thr Ala Asn
Thr Gln Cys His Glu Lys Met Pro Val Lys Asp Ile Tyr 355 360 365Phe
Gln Ser Cys Val Phe Asp Leu Leu Thr Thr Gly Asp Ala Asn Phe 370 375
380Thr Ala Ala Ala His Ser Ala Leu Glu Asp Val Glu Ala Leu His
Pro385 390 395 400Arg Lys Glu Arg Trp His Ile Phe Pro Ser Ser Gly
Asn Gly Thr Pro 405 410 415Arg Gly Gly Ser Asp Leu Ser Val Ser Leu
Gly Leu Thr Cys Leu Ile 420 425 430Leu Ile Val Phe Leu
435241272DNAHomo sapiens 24atgggccagt cccctagtcc caggtcctcc
catggcagtc ccccaactct aagcactctc 60actctcctgc tgctcctctg tggacatgct
cattctcaat gcaagatcct ccgctgcaat 120gctgagtacg tatcgtccac
tctgagcctt agaggtgggg gttcatcagg agcacttcga 180ggaggaggag
gaggaggccg gggtggaggg gtgggctctg gcggcctctg tcgagccctc
240cgctcctatg cgctctgcac tcggcgcacc gcccgcacct gccgcgggga
cctcgccttc 300cattcggcgg tacatggcat cgaagacctg atgatccagc
acaactgctc ccgccagggc 360cctacagccc ctcccccgcc ccggggcccc
gcccttccag gcgcgggctc cggcctccct 420gccccggacc cttgtgacta
tgaaggccgg ttttcccggc
tgcatggtcg tcccccgggg 480ttcttgcatt gcgcttcctt cggggacccc
catgtgcgca gcttccacca tcactttcac 540acatgccgtg tccaaggagc
ttggcctcta ctggataatg acttcctctt tgtccaagcc 600accagctccc
ccatggcgtt gggggccaac gctaccgcca cccggaagct caccatcata
660tttaagaaca tgcaggaatg cattgatcag aaggtgtatc aggctgaggt
ggataatctt 720cctgtagcct ttgaagatgg ttctatcaat ggaggtgacc
gacctggggg atccagtttg 780tcgattcaaa ctgctaaccc tgggaaccat
gtggagatcc aagctgccta cattggcaca 840actataatca ttcggcagac
agctgggcag ctctccttct ccatcaaggt agcagaggat 900gtggccatgg
ccttctcagc tgaacaggac ctgcagctct gtgttggggg gtgccctcca
960agtcagcgac tctctcgatc agagcgcaat cgtcggggag ctataaccat
tgatactgcc 1020agacggctgt gcaaggaagg gcttccagtg gaagatgctt
acttccattc ctgtgtcttt 1080gatgttttaa tttctggtga tcccaacttt
accgtggcag ctcaggcagc actggaggat 1140gcccgagcct tcctgccaga
cttagagaag ctgcatctct tcccctcaga tgctggggtt 1200cctctttcct
cagcaaccct cttagctcca ctcctttctg ggctctttgt tctgtggctt
1260tgcattcagt aa 127225423PRTHomo sapiens 25Met Gly Gln Ser Pro
Ser Pro Arg Ser Ser His Gly Ser Pro Pro Thr 1 5 10 15Leu Ser Thr
Leu Thr Leu Leu Leu Leu Leu Cys Gly His Ala His Ser 20 25 30Gln Cys
Lys Ile Leu Arg Cys Asn Ala Glu Tyr Val Ser Ser Thr Leu 35 40 45Ser
Leu Arg Gly Gly Gly Ser Ser Gly Ala Leu Arg Gly Gly Gly Gly 50 55
60Gly Gly Arg Gly Gly Gly Val Gly Ser Gly Gly Leu Cys Arg Ala Leu
65 70 75 80Arg Ser Tyr Ala Leu Cys Thr Arg Arg Thr Ala Arg Thr Cys
Arg Gly 85 90 95Asp Leu Ala Phe His Ser Ala Val His Gly Ile Glu Asp
Leu Met Ile 100 105 110Gln His Asn Cys Ser Arg Gln Gly Pro Thr Ala
Pro Pro Pro Pro Arg 115 120 125Gly Pro Ala Leu Pro Gly Ala Gly Ser
Gly Leu Pro Ala Pro Asp Pro 130 135 140Cys Asp Tyr Glu Gly Arg Phe
Ser Arg Leu His Gly Arg Pro Pro Gly145 150 155 160Phe Leu His Cys
Ala Ser Phe Gly Asp Pro His Val Arg Ser Phe His 165 170 175His His
Phe His Thr Cys Arg Val Gln Gly Ala Trp Pro Leu Leu Asp 180 185
190Asn Asp Phe Leu Phe Val Gln Ala Thr Ser Ser Pro Met Ala Leu Gly
195 200 205Ala Asn Ala Thr Ala Thr Arg Lys Leu Thr Ile Ile Phe Lys
Asn Met 210 215 220Gln Glu Cys Ile Asp Gln Lys Val Tyr Gln Ala Glu
Val Asp Asn Leu225 230 235 240Pro Val Ala Phe Glu Asp Gly Ser Ile
Asn Gly Gly Asp Arg Pro Gly 245 250 255Gly Ser Ser Leu Ser Ile Gln
Thr Ala Asn Pro Gly Asn His Val Glu 260 265 270Ile Gln Ala Ala Tyr
Ile Gly Thr Thr Ile Ile Ile Arg Gln Thr Ala 275 280 285Gly Gln Leu
Ser Phe Ser Ile Lys Val Ala Glu Asp Val Ala Met Ala 290 295 300Phe
Ser Ala Glu Gln Asp Leu Gln Leu Cys Val Gly Gly Cys Pro Pro305 310
315 320Ser Gln Arg Leu Ser Arg Ser Glu Arg Asn Arg Arg Gly Ala Ile
Thr 325 330 335Ile Asp Thr Ala Arg Arg Leu Cys Lys Glu Gly Leu Pro
Val Glu Asp 340 345 350Ala Tyr Phe His Ser Cys Val Phe Asp Val Leu
Ile Ser Gly Asp Pro 355 360 365Asn Phe Thr Val Ala Ala Gln Ala Ala
Leu Glu Asp Ala Arg Ala Phe 370 375 380Leu Pro Asp Leu Glu Lys Leu
His Leu Phe Pro Ser Asp Ala Gly Val385 390 395 400Pro Leu Ser Ser
Ala Thr Leu Leu Ala Pro Leu Leu Ser Gly Leu Phe 405 410 415Val Leu
Trp Leu Cys Ile Gln 420
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